UA119445C2 - Composition comprising torasemide and baclofen for treating neurological disorders - Google Patents

Abstract

The present invention relates to compositions and methods for the treatment of neurological disorders related to glutamate excitotoxicity and Amyloid T toxicity. More specifically, the present invention relates to novel combinatorial therapies of Multiple Sclerosis, Alzheimer's disease, Alzheimer's disease related disorders, Amyotrophic Lateral Sclerosis, Parkinson's disease, Huntington's disease, neuropathic pain, alcoholic neuropathy, alcoholism or alcohol withdrawal, or spinal cord injury.

Description

The field of technology to which the invention belongs

This invention relates to compositions and methods of treatment of neurological diseases and disorders. More specifically, the present invention relates to new combination therapies for diseases such as Alzheimer's disease and related diseases, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, neuropathy, alcoholism, alcohol withdrawal, Huntington's disease and spinal cord lesions.

Prior art

Alzheimer's disease (AD) is a prototypical cortical dementia, which is characterized by memory deficits together with dysphasia (a speech disorder in which speech and language comprehension are impaired), dyspraxia (inability to coordinate and carry out certain intentional movements and gestures in the absence of motor or sensory insufficiency) and agnosia (ability to recognize subjects, people, sounds, shapes or smells) due to the involvement of cortical association areas (1-4).

AI is currently the most common cause of dementia. Clinically, it is characterized by a global decline in cognitive function that progresses slowly and leaves end-stage patients bedridden, incontinent and dependent on custodial care. Death occurs, on average, 9 years after diagnosis (5).

The incidence rate of AI increases dramatically with age. According to United Nations demographic forecasts, the number of people over 80 will approach 370 million by 2050. Currently, it is estimated that 50 to 95 people over the age of 85 suffer from AI. Therefore, in 50 years, more than 100 million people worldwide will suffer from dementia. The sheer number of people requiring ongoing care and other services will strain medical, financial, and human resources (6). Deterioration of memory is an early sign of the disease and includes episodic memory (memory for day-to-day events).

Semantic memory (memory for verbal and visual content) is affected later in the disease. Pathological hallmarks of AJ include amyloid plaques that contain amyloid-beta (Abreia), neurofibrillary tangles (METs) that contain Tam, and neuronal and synaptic dysfunction and loss (7-9). Over the last decade, two main hypotheses have been proposed regarding the causes of AI:

From the "amyloid cascade hypothesis", which states that the neurodegenerative process is a series of events caused by abnormal processing of amyloid precursor protein (APP) (10), and the "neuronal cytoskeletal degeneration hypothesis" (11), which suggests that cytoskeletal changes are initiating events. The most common theory to explain the progression of Aβ remains the amyloid cascade hypothesis (12-14), and AI researchers are primarily focused on identifying the mechanisms underlying protein-associated toxicity.

Abreia Microvascular permeability and remodeling, aberrant angiogenesis (and the blood-brain barrier) have been identified as key events contributing to APP toxicity in the amyloid cascade (15). In contrast, the Tash protein has received much less attention from the pharmaceutical industry than amyloid, due to fundamental and practical problems.

Moreover, altered synaptic density is a pathological lesion that correlates better with cognitive impairment than the other two.

Studies have shown that amyloid pathology appears to progress in a neurotransmitter-specific manner, with cholinergic terminals appearing to be most vulnerable, followed by glutamatergic terminals, and finally GABAergic terminals (9). Glutamate is the most common excitatory neurotransmitter in the mammalian nervous system. In pathological conditions, its abnormal accumulation in the synaptic cleft leads to hyperactivation of glutamate receptors (16). Abnormal accumulation of glutamate in the synaptic cleft leads to hyperactivation of glutamate receptors, which results in pathological processes and, finally, the death of the neuron. This process, called excitotoxicity, is commonly observed in neuronal tissue during acute and chronic neurological disorders.

It is becoming clear that excitotoxicity is involved in the pathogenesis of several diseases of different etiologies, such as: spinal cord injuries, stroke, traumatic brain injury, hearing loss, alcoholism and alcohol withdrawal, alcoholic neuropathy or neuropathic pain, as well as neurodegenerative diseases such as diffuse multiple sclerosis, Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, and Huntington's disease (17-19). The development of effective treatment for these diseases remains the main problem of public health care due to the frequency of their occurrence, as well as the lack of treatment procedures.

MMOAC antagonists that target different sites of this receptor have been tested to counteract excitotoxicity. Noncompetitive MMOAC antagonists target the ion channel pore, thereby reducing calcium entry into postsynaptic neurons. Some of them have reached approval status. For example, mamantine is currently approved for moderate to severe Alzheimer's disease. It is clinically tested for other indications that include an excitotoxicity component, such as alcohol dependence (Phase II), amyotrophic lateral sclerosis (Phase II), dementia associated with Parkinsonism (Phase II), epilepsy, Huntington's disease (Phase II

MI), multiple sclerosis (MI phase), Parkinson's disease (MI phase) and traumatic brain injury (MI phase

IM). This molecule, however, shows limited benefit in most patients with the disease

Alzheimer's, as it exhibits moderate symptomatic effects. Another approach to limiting excitotoxicity is inhibition of presynaptic glutamate release. Riluzole, currently approved for amyotrophic lateral sclerosis, has shown promising results in ischemia and brain injury models (20-23). It is currently being tested in phase II trials for early-onset multiple sclerosis, Parkinson's disease (showing no better results than placebo), and spinal cord injury. In 1995, the drug achieved orphan drug status for the treatment of amyotrophic lateral sclerosis and in 1996 for the treatment of Huntington's disease.

UMO 2009/133128, MO 2009/133141, MO 2009/133142 and UMO 2011/054759 disclose molecules that can be used in compositions for the treatment of neurological disorders.

Despite active research in this field, there is a need to develop alternative or improved effective therapies for neurological disorders, and in particular, neurological disorders that are associated with glutamate and/or amyloid beta toxicity. Currently, the invention offers new types of treatment for such neurological disorders of the central nervous system (CM5) and peripheral nervous system (PM5).

The essence of the invention

The subject of this invention is to offer new therapeutic approaches to the treatment of neurological disorders.

The invention is due, in particular, to the unexpected discovery by the authors that torasemide, trimetazidine, mexiletine, bromocriptine, ifenprodil and moxifloxacin, individually or in combination, represent new and effective therapies for the treatment of neurological disorders.

The invention therefore provides new compositions and methods for the treatment of neurological disorders, in particular, AO and related disorders, multiple sclerosis (M5), amyotrophic lateral sclerosis

Zo (AG 5), Parkinson's disease (RO), neuropathy (eg, neuropathic pain or alcoholic neuropathy), alcoholism or alcohol withdrawal, peripheral nervous system lesions, Huntington's disease (HO), and spinal cord injury.

In particular, the invention relates to a composition used for the treatment of neurological disorders, which includes at least torasemide, trimetazidine, mexiletine, ifenprodil, moxifloxacin or bromocriptine, or their salts, prodrugs, derivatives or compositions with delayed release.

In addition, the present invention relates to a composition that includes at least one first compound selected from the group consisting of torasemide, trimetazidine, mexiletine, ifenprodil, moxifloxacin and bromocriptine, or a salt, prodrug, derivative of any chemical purity or a composition thereof with delayed release in combination with at least one second compound different from said first compound selected from salts, prodrugs, derivatives of any chemical purity of sulfisoxazole, methimazole, prilocaine, diphylline, quinacrine, carbenoxolone, acamprosate, aminocaproic acid, baclofen, cabergoline, diethylcarbamazine, cinalcet, cinnarizine, epleron, fenoldopam, leflunomide, levosimendan, sulodexide, terminafine, zonisamide, etomidate, phenoformin, trimetazidine, mexiletine, bromocriptine, ifenrodil, torasemide and moxifloxacin, or their compositions with delayed release or for simultaneous, separate introduction.

Another subject of this invention is a composition for use in the treatment of a neurological disorder, which includes at least one first compound selected from the group consisting of torasemide, trimetazidine, mexiletine, ifenprodil, moxifloxacin, bromocriptine, or their salts, prodrugs, derivatives of any chemical purity, or a sustained release composition, in combination with at least one second compound other than said first compound selected from sulfisoxazole, methimazole, prilocaine, diphylline, quinacrine, carbenoxolone, acamprosate, aminocaproic acid, baclofen, cabergoline, diethylcarbamazine, cinacalcet, cinnarizine, eplerenone, fenoldopam, leflunomide, levosimendan, sulodexide, terbinafine, zonisamide, etomidate, phenformin, trimetazidine, mexiletine, bromocriptine, ifenprodil, torasemide, and moxifloxacin, their salts, prodrugs, derivatives of any chemical purity , or compositions for delayed 60 release, for simultaneous, separate or sequential input.

The present invention also relates to a composition that includes at least one first compound selected from the group consisting of torasemide, trimetazidine, mexiletine, ifenprodil, moxifloxacin and bromocriptine, or their salts, prodrugs, a derivative of any chemical purity or a composition of sustained release in combination with at least one second compound other than said first compound selected from sulfisoxazole, methimazole, prilocaine, diphylline, quinacrine, carbenoxolone, acamprosate, aminocaproic acid, baclofen, cabergoline, diethylcarbamazine, cinalcet, cinnarizine, eplerone, fenoldopam , leflunomide, levosimendan, sulodexide, terminafine, zonisamide, etomidate, phenoformin, trimetazidine, mexiletine, bromocriptine, ifenrodil, torasemide and moxifloxacin, or their compositions with delayed release or their salts, prodrugs, derivatives of any chemical purity for simultaneous, separate or sequential input.

The best drug compositions include 1, 2, 3, 4 or 5 different drugs, more preferably 2, C or 4. Moreover, the above drug compositions can also be used in an additional composition with one or more additional drugs or treatments, that have a beneficial effect on subjects with a neurological disorder.

The invention also relates to a method of treating a neurological disorder, which involves administering to a subject in need of it the medicinal product or composition specified above.

Another subject of the invention relates to a method of treating a neurological disorder, which involves the simultaneous, separate or sequential administration of a combination of medicinal substances described above to a subject in need thereof.

Another object of the invention relates to the use of at least one compound selected from the group consisting of torasemide, trimetazimide, mexiletine, ifenprodil, bromocriptine and moxifloxacin, or their salt(s), prodrugs, derivative(s) of any of chemical purity, or composition(s) for sustained release, for the manufacture of a medicinal product for the treatment of a neurological disorder.

Another subject of the invention relates to the use of combinations of drugs, described above, for the manufacture of a drug for the treatment of a neurological disorder.

The invention can be used for any subject - a mammal, especially for a subject - a person, at any stage of the disease.

Zo Brief description of the drawings

For Figures 1-27, "7 p"0.05: significantly different from the control (without intoxication); "p5": insignificant effect (AMOMA « Dunnett's a posteriori test)

Figure 1: The influence of pretreatment with selected drugs on the damage of НВМЕС by human AB:-422. A) Verification of the experimental model used for drug screening: 1 hour. pre-treatment with MESE at 10 nM significantly protects the capillary network from this amyloid lesion (1470 95 capillary network in comparison with amyloid intoxication). Torasemide (B) and bromocriptine (C) in doses up to 400 nM and 3.2 nM significantly prevent intoxication, respectively, while no effect or a weak effect is noted for the upper and lower doses. 9: p«e0.05: significantly different from amyloid intoxication.

Figure 2: Effect of pretreatment with selected drugs on the release of IOH in AVi-2 toxicity tests on rat primary cortical cells. A) Verification of the experimental model used for drug screening: 1 hour. pretreatment with estradiol (150 ng/ml) significantly protected neurons from amyloid damage (-70 Ob), which is considered as a positive control for neuroprotection. For all experiments, AB:-42 produced significant intoxication compared to vehicle-treated neurons. Intoxication is significantly inhibited by bromocriptine (40 nM, -29 95) (B), trimetazidine (40 nM, -94 Ov) (C), ifenprodil (600 nM, -94 95) (0), mexiletine (3.2 NM, - 73 90) (E), moxifloxacin (20 nM, -63 Fo) (P). It is worth noting that at other drug concentrations, the effect is absent or a weaker effect is noted for upper or lower doses. b: p«0.05: significantly different from AB'-42 intoxication.

Figure 3: The effect of the combination of baclofen and torasemide on the total length of the capillary network in HVMES cultures intoxicated with beta-amyloid. Aggregated human amyloid peptide (AVi-a2 2.5 NM) produced significant intoxication, greater than 40 95, compared to vehicle-treated cells. This intoxication is significantly inhibited by the combination of baclofen and torasemide (A), while at these concentrations, baclofen (B) and torasemide (C) separately do not show a significant effect on intoxication. 9: p«e0.05, significantly different from AD -a2 intoxication.

Figure 4: Effect of combination therapy with sulfisoxazole and torasemide on total capillary network length in β-amyloid-intoxicated HVMES cultures. Aggregated human amyloid peptide (AVi-a2 2.5 IU) produced significant intoxication, above 40 90, compared to vehicle-treated bo cells. This intoxication is significantly inhibited by the combination of sulfisoxazole and torasemide (A), while at these concentrations, sulfisoxazole (B) and torasemide (C) individually do not have a significant effect on intoxication. Ф: p«0.05, significantly different from AVDch1-42 intoxication.

Figure 5: Effect of combination therapy with eplerenone and torasemide on total capillary network length in β-amyloid-intoxicated HVMES cultures. Aggregated human amyloid peptide (AVi-a2 2.5 IU) produced significant intoxication, above 40 90, compared to cells treated with the vehicle. This intoxication is significantly inhibited by the combination of eplerenone and torasemide (A), while at these concentrations, torasemide (B) and eplerenone (C) separately do not show a significant effect on intoxication. b: p«0.05, significantly different from AB-22 intoxication.

Figure 6: Effect of bromocriptine and sulfisoxazole combination therapy on total capillary network length in beta-amyloid-intoxicated HVMES cultures. Aggregated human amyloid peptide (AB:-42 2.5 NM) produced significant toxicity, above 40,905, compared to vehicle-treated cells. This intoxication is significantly inhibited by the combination of bromocriptine and sulfisoxazole (A), while at these concentrations, bromocriptine (B) and sulfisoxazole (C) individually do not have a significant effect on intoxication. 9: p«e0.05, significantly different from AB'1-42 intoxication.

Figure 7: Effect of combined therapy with acamprosate and ifenprodil on ION release in ADi-42 toxicity tests on rat primary cortical cells. Aggregated human amyloid peptide (ADi-42 10 MM) produced significant intoxication compared to neurons treated with the vehicle. This intoxication is significantly inhibited by the combination of acamprosate and ifenprodil (A), while at these concentrations acamprosate (B) and ifenprodil (C) individually do not show a significant effect on intoxication. 2: p«0.05, significantly different from intoxication

AV:-a".

Figure 8: Effects of combined baclofen and mexiletine therapy on ION release in ADi-42 toxicity tests on rat primary cortical cells. Aggregated human amyloid peptide (ADi-42 10 MM) produced significant intoxication compared to neurons treated with the vehicle. This intoxication is significantly inhibited by the combination of baclofen and mexiletin (A), while at these concentrations baclofen (B) and myxiletin (C) separately are not detected

From a significant effect on intoxication. 9: p-0.051, different from AB-22 intoxication.

Figure 9: Effect of combined therapy with baclofen and trimetazidine on GON release in ADi-42 toxicity tests on rat primary cortical cells. Aggregated human amyloid peptide (ADi-42 10 MM) produced significant intoxication compared to neurons treated with the vehicle. This intoxication is significantly inhibited by the combination of baclofen and trimetazidine (A), while at these concentrations, baclofen (B) and trimetazidine (C) individually do not show a significant effect on intoxication. 2: p«0.05, significantly different from intoxication

AV:-a".

Figure 10: Effect of combined therapy with cinacalcet and mexiletine on GH OH release in ADi-42 toxicity tests on rat primary cortical cells. Aggregated human amyloid peptide (ADi-42 10 MM) produced significant intoxication compared to neurons treated with the vehicle. This intoxication is significantly inhibited by the combination of cinacalcet and mexiletin (A), while in these concentrations cinacalcet (B) and myxiletin (C) separately do not show a significant effect on intoxication. 2: p«0.05, significantly different from intoxication

AV:-a".

Figure 11: Effect of combined therapy with tyrarizine and trimetazidine on ION release in ADi-42 toxicity tests on rat primary cortical cells. Aggregated human amyloid peptide (ADi-42 10 NM) produces significant intoxication, compared to neurons treated with the carrier. This intoxication is significantly inhibited by the combination of cinnarizine and trimetazidine (A), while in these concentrations cinnarizine (B) and trimetazidine (C) separately do not show a significant effect on intoxication. 2: p«0.05, significantly different from intoxication

AV:-a".

Figure 12: Effect of combined therapy with trimetazidine and zonisamide on GH OH release in ADi-42 toxicity tests on rat primary cortical cells. Aggregated human amyloid peptide (ADi-42 10 NM) produces significant intoxication, compared to neurons treated with the carrier. This intoxication is significantly inhibited by the combination of trimetazidine and zonisamide (A), while at these concentrations trimetazidine (B) and zonisamide (C) individually do not show a significant effect on intoxication. 9: p«e0.05, significantly different from AB intoxication: -a2.

Figure 13: Effect of combination therapy with terminafine and torasemide on the total length of the capillary network in cultures of HVMES intoxicated with beta-amyloid. Aggregated human 60 amyloid peptide (AVi-42 2.5 MM) produced significant toxicity, above 4095, compared to vehicle-treated cells. This intoxication is significantly inhibited by the combination of terbinafine and torasemide (A), while at these concentrations terbinafine (B) and torasemide (C) separately do not show a significant effect on intoxication. b: p«0.05, significantly different from AB-22 intoxication.

Figure 14: Effect of combined therapy with cinacalcet and mexiletine on the total length of the capillary network in HVMES cultures intoxicated with beta-amyloid. Aggregated human amyloid peptide (AVi-42 2.5 MM) produced significant toxicity, above 4095, compared to vehicle-treated cells. This intoxication is significantly inhibited by the combination of cinacalcet and mexiletine (A), while at these concentrations cinacalcet (B) and mexiletine (C) separately do not show a significant effect on intoxication. b: p«0.05, significantly different from AB-22 intoxication.

Figure 15: Effects of combined baclofen and torasemide therapy on ION release in ADi-42 toxicity tests on rat primary cortical cells. Aggregated human amyloid peptide (ADi-42 10 MM) produced significant intoxication compared to neurons treated with the vehicle. This intoxication is significantly inhibited by the combination of baclofen and torasemide (A), while at these concentrations, baclofen (B) and torasemide (C) separately do not show a significant effect on intoxication. 9: p«e0.0572, significantly different from AB:-42 intoxication.

Figure 16: Effect of combined therapy with acamprosate and ifenprodil on GON release in ADi-42 toxicity tests on rat primary cortical cells. Aggregated human amyloid peptide (ADi-42 10 MM) produced significant intoxication compared to neurons treated with the vehicle. This intoxication is significantly inhibited by the combination of sulfisoxazole and torasemide (A), while at these concentrations, torasemide (B) and sulfisoxazole (C) individually do not have a significant effect on intoxication. 2: p«0.05, significantly different from intoxication

AV:-a".

Figure 17: Effect of combination therapy with moxifloxacin and trimetazidine on release

ION in AVi--g toxicity tests on rat primary cortical cells. Aggregated human amyloid peptide (AB:-42 10 NM) produces significant intoxication, compared to neurons treated with the carrier. This intoxication is significantly inhibited by the combination of moxifloxacin and trimetazidine (A). Addition of moxifloxacin allows to increase by 100 95 the effect which

Zo is observed for trimetazidan (C) alone, while, at the same concentration, moxifloxacin (B) alone does not reveal a significant effect on intoxication. 9: p«e0.05, significantly different from AB'1-42 intoxication.

Figure 18: Effect of combined therapy with moxifloxacin and baclofen on the release of OH in AB:1-42 toxicity tests on rat primary cortical cells. Aggregated human amyloid peptide (ADi-42 10 MM) produced significant intoxication compared to neurons treated with the vehicle. This intoxication is significantly inhibited by the combination of moxifloxacin and baclofen (A), while at these concentrations, moxifloxacin (B) and baclofen (C) individually do not have a significant effect on intoxication. 2: p«0.05, significantly different from intoxication

AV:-a".

Figure 19: Effect of combination therapy with moxifloxacin and cinacalcet on release

ION in AVi--g toxicity tests on rat primary cortical cells. Aggregated human amyloid peptide (AB:-42 10 NM) produces significant intoxication, compared to neurons treated with the carrier. This intoxication is significantly inhibited by the combination of moxifloxacin and cinacalcet (A), while at these concentrations moxifloxacin (B) and cinacalcet (C) separately do not show a significant effect on intoxication. 2: p«0.05, significantly different from intoxication

AV:-a".

Figure 20: Effect of combined therapy with moxifloxacin and zonisamide on the release of OH in AB:1-42 toxicity tests on rat primary cortical cells. Aggregated human amyloid peptide (ADi-42 10 MM) produced significant intoxication compared to neurons treated with the vehicle. This intoxication is significantly inhibited by the combination of moxifloxacin and zonisamide (A). The addition of moxifloxacin increases by 8195 the effect observed for zonisamide (C) alone, while, at the same concentration, moxifloxacin (B) alone does not show a significant effect on intoxication. 9: p«e0.05, significantly different from AVD-22 intoxication.

Figure 21: The effect of combined therapy with moxifloxacin and sulfisoxazole on the release of GON in AVi--g toxicity tests on rat primary cortical cells.

Aggregated human amyloid peptide (AB1-42 10 NM) produced significant intoxication compared to neurons treated with vehicle. This intoxication is significantly inhibited by the combination of moxifloxacin and sulfisoxazole (A), while at these concentrations, moxifloxacin (B) and sulfisoxazole (C) alone do not have a significant effect on intoxication. 9: p«e0.05, significantly different from AB'1-42 intoxication.

Figure 22: Effect of combined therapy with mexiletine (MEX) and ifenprodil (IEM) on the release of TON in AVi-g toxicity tests on rat primary cortical cells.

Aggregated human amyloid peptide (AB:1-42 10 NM) produced significant intoxication compared to neurons treated with the vehicle. This intoxication is significantly inhibited by the combination of 25.6 PM mexiletine and 24 nM ifenprodil, while at these concentrations mexiletine and ifenprodil alone do not show a significant effect on intoxication. Ф: p«0.0572, significantly different from AB-22 intoxication.

Figure 23: Effects of the combination of baclofen (ALL) and torasemide (TOC) on total neurite network length in beta-amyloid-intoxicated cortical neurons. Human amyloid peptide (AVi-4a2 2.5 μM) produced significant intoxication, above 1595, compared to vehicle-treated cells. This intoxication is significantly inhibited by the combination of baclofen and torasemide (A); moreover, this combination makes it possible to increase the growth of neurites. 9: p«e0.05, significantly different from AB-42 intoxication.

Figure 24: Effect of cinacalcet and mexiletine combination therapy against glutamate toxicity on neuronal cortical cells. Glutamate intoxication is significantly inhibited by the combination of cinocalcet (64 pM) and mexiletine (25.6 pM), while at these concentrations, cinacalcet and mexiletine alone have no significant effect on intoxication. 9: p«0.001, significantly different from glutamate intoxication; (AMOMA "k post-posterior Danet's criterion).

Figure 25: Effect of combination therapy with sulfisoxazole and torasemide against glutamate toxicity on neuronal cortical cells. Glutamate intoxication is significantly inhibited by the combination of sulfisoxazole (64 pM) and torasemide (25.6 pM), while at these concentrations, sulfisoxazole and torasemide alone do not have a significant effect on intoxication. 9: p«0.001, significantly different from glutamate intoxication; (AMOMA "k post-posterior Danet's criterion).

Figure 26: Effect of pretreatment with torasemide (TOC) on ION release in ADVi-42 toxicity tests on rat primary cortical cells. AB:1-42 leads to significant intoxication compared to neurons treated with the vehicle. Intoxication is significantly inhibited by torasemide (200 nM, -9095). 2: p«0.0001: significantly different from AVch-42 intoxication.

Figure 27: Effect of pre-treatment with acamprosate and its derivative homotaurine on the release of IOH in the AB1-4g toxicity tests on rat primary cortical cells. AvVch1-42 leads to significant intoxication compared to neurons treated with the vehicle. Intoxication is equally significantly inhibited by homotaurine and acamprosate (9995, 8 nM). 0: p-0.0001: significantly different from AB-42 intoxication.

Figure 28: Effect of the combination of baclofen (ALL) and torasemide (TOC) on the total length of the neurite network of cortical neurons cultured in the absence of the toxic substance.

An increase in the length of the neurite network is observed when a combination of baclofen (400 n m ) and torasemide (80 n m ) is added to the culture medium; moreover, this combination enhances neurite outgrowth, whereas at these concentrations, baclofen and torasemide alone have no significant effect (p5) on neurite network length. "p" 0.005, significantly different from the control.

Figure 29: Effects of the combination of baclofen (ALL) and torasemide (TOC) on the stimulation of nerve regeneration after nerve compression. A-A animals subjected to nerve injury (nerve crush) treated with the combination of baclofen and torasemide show a significantly lower SMAP latency when stimulating the injured sciatic nerve on day 7 and day 30 from the moment of nerve crush compared to sham-operated animals (white bar ) or animals treated with a carrier. B-B signal amplitudes of muscle evoked potentials during stimulation of the sciatic nerve are lower in animals subjected to nerve injury compared to sham-operated animals both on day 7 and on day 30 after nerve compression.

A significant increase in SMAR amplitude is observed on the 30th day after nerve crush for animals treated with baclofen (3 mg/kg) - torasemide (400 μg/kg) (dose 3). "р" 0.05; yakr-"0.001; хр" 0.0001, significantly different from animals treated with the carrier (black column).

Detailed description of the invention

The present invention provides novel compositions for the treatment of neurological disorders.

The invention describes a new application of drugs or new combinations of drugs that can effectively correct such diseases and can be used to treat patients.

The invention provides the treatment of any neurological disorder, whether of the central or peripheral system, in particular, disorders in which excitotoxicity of amyloid and glutamate is involved. Specific examples of such disorders include neurodegenerative diseases such as Alzheimer's disease and related diseases, multiple sclerosis (M5), amyotrophic lateral sclerosis (AG5), Parkinson's disease (PD), Huntington's disease (HO), or other neurological disorders such as neuropathies ( eg alcoholic neuropathy or neuropathic pain), alcoholism or alcohol withdrawal and spinal cord injury. "Neuropathies" refer to conditions in which the nerves of the peripheral nervous system are damaged, including peripheral nervous system damage caused by genetic factors, inflammatory disease, chemical damage, including drugs (vincristine, oxaliplatin, ethyl alcohol), or direct physical insult to the nerve. Treatment of neuropathies also involves treatment of neuropathic pain.

Damage to the peripheral nervous system can be classified according to the stage of the neuronal stroke. The invention is suitable for the treatment of nerve lesions ranging from neuropraxia (a condition in which only the signaling ability of the nerve is affected), to axonotmesis (a lesion that touches the axons without damage to the surrounding connective tissues of the nerves), as well as neurotmesis (a lesion in which the axon suffers , as well as surrounding tissues).

Changes in the axon or surrounding tissues (such as myelin) may have a genetic origin.

An example of hereditary neuropathies is the so-called family of Charcot-Marie-Tooth diseases.

Charcot-Marie-Tooth disease is a progressive disorder affecting peripheral nerves characterized by an alteration in a specific gene(s). The mutation(s) results in damage to axons, which transmit nerve impulses, and/or affects the production of myelin sheath by Schwann cells, which affects the speed of nerve impulse transmission. They are represented by several types (categorized according to the function of clinical signs) and subtypes

CMT (according to genetic classification). SMT types are SMT1, SMT2, SMT3Z, SMT4, SMT5,

SMT6, SMTOI, SMTKI, SMIKH. Related peripheral neuropathies are, for example, NMR (hereditary neuropathy with a tendency to paralysis from nerve compression), severe demyelinating neuropathies (055, Dezherin-Sottas syndrome), SNM (congenital hypomyelinating neuropathy).

About 70,905 patients have SMT (dymyelizing type) and SMT2 (axonal type)

CMT

The invention is particularly suitable for the treatment of AO and related disorders. In the context of this

According to the invention, the term "disorder associated with AI" includes senile dementia of the AI type (5BOA(ZOAT), Lewy body dementia, vascular dementia, mild cognitive impairment (MC) and age-related memory impairment (AAMI).

The term "treatment" as used herein includes the therapy, prevention, prophylaxis, retardation or reduction of symptoms caused by or caused by the aforementioned diseases or disorders. The term treatment includes specific control of disease progression and associated disorders. The term treatment in particular includes ii) protection against amyloid-beta-induced toxicity, or reduction or retardation of said toxicity, and/or ii) protection against glutamate excitotoxicity, or reduction or retardation of said toxicity in subjects subject to treatment. The term treatment also refers to the improvement of a cognitive symptom or the protection of neuronal cells. In relation to neuropathies, the term treatment also includes nerve regeneration, which includes remyelination, the formation of new neurons, glia, axons, myelin or synapses.

In the context of the invention, the designation of specific compounds should include not only the specifically labeled molecules, but also any pharmaceutically acceptable salt, hydrate, derivative (eg, ester, simple ester), isomer, racemate, conjugate, or prodrug thereof of any degree of purity.

The term "prodrug", as used herein, refers to any functional derivative (or precursor) of a compound of the present invention that, when introduced into a biological system, generates said compound as a result of, for example, a spontaneous chemical reaction(s) ), enzyme-catalyzed chemical reaction(s), and/or metabolic chemical reaction(s). Prodrugs, as a rule, are inactive or less active than the resulting drug and can be used, for example, to improve the physicochemical properties of the drug, to direct the drug to a specific tissue, to improve the pharmacokinetic and pharmacodynamic properties of the drug, and /(or to reduce unwanted side effects. Prodrugs, as a rule, have a structure

X-drug, where X is a component of the inert carrier, and the drug is an active compound, the prodrug is less active than the drug, and the drug is released from the carrier ip mimo. Some of the common functional groups that are often included in the development of prodrugs include, in particular, a carboxyl, hydroxyl, amino, phosphate/phosphonate, and carbonyl group. Prodrugs typically prepared by modification of these groups include, but are not limited to, esters, carbonates, carbamates, amides, and phosphates.

Specific technical recommendations for the selection of suitable prodrugs are known from the field of general knowledge (24-28). In addition, the preparation of prodrugs can be carried out using conventional methods known to those skilled in the art. Methods that can be used for the synthesis of prodrugs are described in numerous reviews on this topic (25; 29-35). For example, arbaclofen placarbil is listed in the Spatial Research Database (http://www.spet.5i5.pit.pipy.dom/spetiaritsi5/), and arbaclofen placarbil are well-known prodrugs of baclofen (36; 43).

The term "derivative" of a compound includes any molecule that is functionally or structurally related to said compound, such as an acid, amide, ester, simple ether, acetylated variant, hydroxylated variant or alkylated (C1-Cb) variant of such compound. The term derivative also includes a structurally related compound with the loss of one or more of the substituents listed above. For example, homotaurine is a deacetylated derivative of acamprosate. Preferred derivative compounds are molecules that have a significant degree of similarity to the specified compound, as determined by known methods. Similar compounds, along with their index of similarity to the parent molecule, can be found in numerous databases, such as Ryrspet (per-/ribspet.pebi.pit.piy.dom/5zeagsp/) or ЮОгидрапк (пер:/Lumly.agidrapk.sa/ ). In a preferred embodiment, the derivatives should have a Tanimoto similarity index higher than 0.4, preferably higher than 0.6, even better, higher than 0.7, to the parent drug. The Tanimoto similarity index is widely used to measure the structural similarity between two molecules. The Tanimoto similarity index can be calculated using software such as ZtaiYi MoiIesshe Zyrodgarp Oeiesiog (37-38), available online (pPEr:/Limly.eri.as.iklApogpiop-5gm/5opmage/5M50O0/). Preferably, the derivatives should be related both structurally and functionally to the parent compounds, that is, they should also retain at least part of the activity of the parent drug, preferably they should show protective activity against AB and glutamate toxicity.

The term derivative also includes metabolites of a drug, for example, a molecule that is obtained as a result of (biochemical) modification(s) or processing of said drug after administration to the body, usually with the help of specialized enzymatic systems, and which demonstrate or retain the biological activity of the drug tool Metabolites

Zo have been described as being responsible for most of the therapeutic action of the parent drug. In a specific embodiment, the term "metabolite" as used herein refers to a modified or processed drug that retains at least some of the activity of the parent drug, preferably that has protective activity against AB or glutamate toxicity. Examples of metabolites include hydroxylated forms of torasemide resulting from hepatic metabolism of the drug (Hyd Bapk dagabvaze (39).

The term "salt" refers to a pharmaceutically acceptable and relatively non-toxic inorganic or organic acid addition salt of a compound of the present invention. A pharmaceutical salt includes a pair of an acidic, basic, or zwitterionic drug molecule with a counterion to create a drug salt. A wide range of chemical samples can be used in the neutralization reaction. Pharmaceutically acceptable salts according to the invention thus include those obtained by reacting the parent compound, which acts as a base, with an inorganic or organic acid to form a salt, for example, salts of acetic acid, nitric acid, tartaric acid, hydrochloric acid, sulfuric acid , phosphoric acid, methanesulfonic acid, camphorsulfonic acid, oxalic acid, maleic acid, succinic acid or citric acid. Pharmaceutically acceptable salts of the present invention also include those in which the parent compound functions as an acid and reacts with a suitable base to form salts, for example sodium, potassium, calcium, magnesium, ammonium or choline. Although most of these active substances are bioequivalent, some may, among other things, have properties of increased solubility or bioavailability. The selection of salt is currently a standard operation in the process of developing a medicinal product, which was disclosed by N. 5ayi and S. Umeptiiy in their instructions (40).

The term "combination" or "combined treatment/therapy" means a treatment in which at least two or more drugs are simultaneously administered to a subject to achieve a biological effect. In the combined therapy according to this invention, at least two drugs can be administered together or separately, simultaneously or sequentially. Also, at least two drugs can be administered by different routes and using different protocols. Thus, although the drugs of the combination can be combined together, they can also be used separately. 60 As described in the examples, torasemide, trimetazidine, mexiletine, ifenprodil, bromocriptine, and moxifloxacin have unexpectedly strong effects on biological processes involved in neurological disorders. Moreover, these compounds have demonstrated a very effective ability to correct the symptoms of such compounds. These molecules, alone or in combination therapies, therefore represent new approaches to the treatment of neurological disorders such as

Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, disease

Huntington's disease, neuropathies (such as neuropathic pain or alcoholic neuropathy), alcoholism or alcohol withdrawal, or spinal cord injury. Combinations of these drugs with other selected compounds (see Table 2) are particularly advantageous in that they produce surprising and unexpected synergistic effects at doses in which the drugs alone have no effect. Also, due to their effectiveness, this document describes drug combinations that can be used at lower doses, which is also an additional very significant advantage.

In this regard, in a specific embodiment, the invention relates to a composition for use in the treatment of AI, disorders associated with AI, M5, RO, AI 5, NO, neuropathies (for example, neuropathic pain or alcoholic neuropathy), alcoholism or alcohol withdrawal, or spinal cord injury, including at least torasemide, trimetazidine, mesquiletine, ifenprodil, bromocriptine, or moxifloxacin, or their salts, prodrugs, derivatives, or sustained release compositions.

Specific CA5Z numbers for each of these compounds are provided in Table 1 below. Table 1 also lists, in particular, standard salts, racemates, prodrugs, metabolites or derivatives of these compounds used in the compositions of the invention.

Table 1

Tanimoto oMexiletine 77777711 17111 Z1828-71-425370-014.. | :K« KS glucuronide oSulfisoxazole. 00000000 127-69-5:4299-609 oTsinacalceto/////7770 | 226256-56-0:364782-343 | g g o Torasemd.//////7777777 | 56211-40-6072810-594.Y | -:/.:f.:DeS (Carboxytarosemid. 77717771 | Metabol./://.//|Н|Н o Bromocriptine 00100 25614-03-3;22260-6141|771111 (Ifenprodil. 77777707 23210-56-2 :23210-568-4.D | :IK (KS

These molecules can be used alone or, preferably, in combination therapy to provide the most effective clinical benefit. In this regard, in the best embodiment, the invention relates to a composition for use in the treatment of a neurological disorder, preferably A, related to AYU disorder, M5, RO, A! 5, NO, neuropathies (eg, neuropathic pain or alcoholic neuropathy), alcoholism or alcohol withdrawal, or spinal cord injury involving any one of the above compounds in combination with at least one other compound selected from sulfisoxazole, methimazole ,

prilocaine, diphylline, quinacrine, carbenoxolone, acamprosate, aminocaproic acid, baclofen, cabergoline, diethylcarbamazine, cinacalcet, cinnarizine, eplerenone, fenoldopam, leflunomide, levosimendan, sulodexide, terbinafine, zonisamide, etomidate, phenformin, trimetazidine, mexiletine, moxiflutine, and bromofendiloxane or torasemide or their salts, prodrugs, derivatives or sustained-release forms.

The specific CA numbers for each of these additional different compounds other than those of Table 1 are presented in Table 2 below:

Table 2 55-3; 847353-30-4

Specific examples of baclofen prodrugs are presented in Napaia et al., 2011 (41), where baclofen esters and carbamic acid and baclofen esters are demonstrated as particularly interesting means of targeted effects on the CNS. Therefore, such prodrugs are particularly suitable for compositions proposed by the invention. Arbaclofen placarbil mentioned earlier are also well known prodrugs and thus can be used instead of baclofen in the compositions of the invention. Other bislofen prots can be detected in the following patent applications: MO 2010102071, 05 2009197958, UMO 2009096985, MO 2009061934, MO 2008086492, 05 2009216037, 20050697

Застосовувані проліки акампросату, такі як складний ефір пантоєвої кислоти й неопентилсульфонілові ефіри, неопентилсульфоніл ефірні проліки або захищені карбоксилатом неопентил сульфоніл ефірні проліки акампросату наведені, зокрема, в МО 2009033069, УМО 2009033061, УМО 2009033054 М/О 2009052191, МО 2009033079, 05 2009/0099255 , 05 2009/0069419, 5 2009/0082464, 05 2009/0082440, and 05 2009/0076147.

In the best embodiment, the invention relates to a composition that includes: - at least one first compound selected from torasemide, trimetazidine, mexiletine, ifenprodil, bromocriptine and moxifloxacin, salt(s), prodrugs, derivative(s) of any chemical purity , or composition(s) of sustained release, in combination with - at least one second compound, different from the specified first compound, selected from sulfisoxazole, methimazole, prilocaine, diphylline, quinacrine, carbenoxolone,

Of acamprosate, aminocaproic acid, baclofen, cabergoline, diethylcarbamazine, cinacalcet, cinnarizine, epleron, fenoldopam, leflunomide, levosimendan, sulodexide, terbinafine, zonisamide, etomidate, phenformin, trimetazidine, mexiletine,

bromocriptine, ifenprodil, toresamide and moxifloxacin, their salt(s), prodrugs, derivative(s), of any chemical purity, or sustained release formulation(s), for use in the treatment of a neurological disorder in a subject in need thereof .

In a specific embodiment, the invention relates to the use of these drugs or compositions for the treatment of AB and related disorders in a subject in need thereof.

In a specific application, the invention relates to the use of these drugs or compositions for the treatment of "M5, PO, AI5, NO, neuropathies (eg, neuropathic pain or alcoholic neuropathy), alcoholism or alcohol withdrawal, or spinal cord injury in a subject, who needs it.

As described in the examples, combined therapy with the use of one or more of the above drugs leads to effective correction of the disease

Alzheimer's and other neurological diseases. As illustrated in the experimental section, compositions that include at least torasemide, trimetazidine, mexiletine, ifenprodil, bromocriptine and moxifloxacin provide a significant therapeutic and biological effect to prevent the toxic effects of amyloid (AB) protein or peptide on human cells. Moreover, these compositions lead to the improvement of cognitive symptoms, as well as to the inhibition of molecular pathways switched by ArB intoxication within the limits of glutamate excitotoxicity. Therefore, they represent new and powerful ways to treat such a disease.

The experimental section further demonstrates that the aforementioned compositions are also effective i) in synergistically protecting ip migo neuronal cells from glutamate toxicity, and ii) in addition to clinical benefit in ip mimo models for diseases associated with glutamate excitotoxicity.

Preferably, the compositions of drugs proposed by the invention may include 1, 2, 3, 4 or 5 different drugs, even better 2, 3 or 4 different drugs for the combined treatment of Alzheimer's disease, associated with AJ disorders, M5, PO, AG 5, NO, neuropathies (eg, neuropathic pain or alcoholic neuropathy), alcoholism or alcohol withdrawal, or spinal cord injury in a subject in need thereof. In the best embodiment, the medicinal products proposed by the invention are used in combination(s)

Co) for combined separate or sequential input, in order to ensure the most effective impact.

In a specific embodiment, the composition includes (i) torasemide and (its) compounds selected from bromocriptine, baclofen, sulfisoxazole, epleron or terbinafine, or salts, prodrugs, derivatives, or compositions for the sustained release of said compounds (ii) and (ii).

In another specific embodiment, the compositions include (i) trimetazidine and (ii) compounds selected from baclofen, cinnarizine, zonisamide or moxifloxacin, or salts, prodrugs, derivatives or compositions for the delayed release of said compounds (ii) and (ii).

According to an additional specific embodiment, the composition includes (i) moxifloxacin and (ii) compounds selected from baclofen, cinacalcet, zonisamide, sulfisoxazole or trimetazidine, or salts, prodrugs, derivatives, or compositions for sustained release of said compounds (ii) and (ii) ).

In another additional specific embodiment, the composition includes (ii) mexiletine and (ii) compounds selected from baclofen, cinacalcet, ifenprodil, or levosimendan or salts, prodrugs, derivatives, or sustained release compositions of said compound (ii) and (ii).

A specific embodiment also relates to a composition that includes (ii) ifenprodil and (ii) compounds selected from acamprosate, levosimendan or mexiletine, or salts, prodrugs, derivatives or sustained release compositions of said compounds (I) and (II).

Preferably, a compound provided by the invention is for use in the treatment of a neurological disorder such as Alzheimer's disease (AD), AJ-related disorder, M5, PO, AG5, NO, neuropathy (eg, neuropathic pain or alcoholic neuropathy), alcoholism or alcohol withdrawal , or damage to the spinal cord, includes one of the following drug combinations, for combined, separate or sequential administration: - baclofen and torasemide; - eplerenone and torasemide; - acamprosate and ifenprodil; - baclofen and mexiletine; - baclofen and trimetazidine; - bromocriptine and sulfisoxazole; - cinacalcet and mexiletine; (516) - cinnarizine and trimetazidine;

- sulfisoxazole and torasemide; - trimetazidine and zonisamide; - levosimendan and mexiletine; - levosimendan and ifenprodil; - levosimendan and trimetazidine; - levosimendan and moxifloxacin; - terbinafine and torasemide; - moxifloxacin and trimetazidine; - moxifloxacin and baclofen; - moxifloxacin and cinacalcet; - moxifloxacin and zonisamide; - moxifloxacin and sulfisoxazole, or - mexiletine and ifenprodil.

Examples of the best compositions proposed by the invention, which include a combination of at least three compounds, for combined, separate or separate administration, are offered below: - baclofen and trimetazidine and torasemide; - baclofen and cinacalcet and torasemide; - baclofen and acamprosate and torasemide; - levosimendan and baclofen and trimetazidine; - levosimendan and aminocaproic acid and trimetazidine; - levosimendan and terbinafine and trimetazidine, or - levosimendan and sulfisoxazole and trimetazidine.

Examples of the best compositions proposed by the invention, which include a combination of at least four compounds, for combined, separate or separate administration, are offered below: - sulfisoxazole, trimetazidine, torasemide and zonisamide; - sulfisoxazole, mexiletine, torasemide and cinacalcet; - baclofen, acamprosate, torasemide and diethylcarbamazine, or

Zo - baclofen, acamprosate, torasemide and ifenprodil.

As described in the experimental section, the above combined therapies proposed by the present invention induce a strong neuroprotective effect against AB toxicity and have a positive effect on behavioral performance and biochemical tests ip mimo. The results demonstrate that the compositions proposed by the invention i) effectively correct molecular pathways switched, i.e., by AV aggregates and i) lead to improvement of neurophysiological lesions observed in sick animals in the form of neuronal survival or synaptic integrity.

Moreover, the presented results also demonstrate that the above combined therapies have an important synergistic neuroprotective effect against glutamate excitotoxicity (Figures 24 and 25, Table 8), the pathway of which is involved in various neurological diseases, such as AYU, M5, PO, AG5, HO , neuropathies (eg, neuropathic pain or alcoholic neuropathy), alcoholism or alcohol withdrawal, or spinal cord injury.

These therapies produce positive results in ip mimo or ip migo models for these diseases.

In addition, the IP MIMO results also demonstrate that the compositions proposed by this invention effectively restore the integrity of the blood-brain barrier, which is known to be affected by some neurological diseases.

The subject of the invention, therefore, are also the compositions defined above for the treatment of a neurological disorder, such as Alzheimer's disease (AD), an AJ-related disorder, M5,

PO, AG 5, NO, neuropathies (eg, neuropathic pain or alcoholic neuropathy), alcoholism or alcohol withdrawal, or spinal cord injury.

Another subject of the invention is the use of the composition defined above for the treatment of a neurological disorder, such as Alzheimer's disease (AD), an AJ-related disorder, M5,

PO, AI 5, NO, neuropathy (for example, neuropathic pain or alcoholic neuropathy), alcoholism or alcohol withdrawal, or spinal cord injury.

The invention also provides a method of treating a neurological disorder such as a disease

Alzheimer's disease (AD), AI-related disorders, M5, PO, AI5, NO, neuropathies (eg, neuropathic pain or alcoholic neuropathy), alcoholism or alcohol withdrawal, or spinal cord injury involving administration to a subject who has requires an effective amount of the composition described above. As previously stated, the compounds in the combination therapy or compositions of the present invention may be formulated together or separately, and administered together, separately or sequentially and/or cyclically.

In this regard, the specific task of the invention is a method of treating Ab associated with

AO disorder, M5, RO, AG 5, NO, neuropathies (eg, neuropathic pain or alcoholic neuropathy), alcoholism or alcohol withdrawal, or spinal cord injury that involves the simultaneous, separate, or sequential administration to a subject in need of an effective amount of the composition described above.

In a preferred embodiment, the invention relates to a method of treating Alzheimer's disease (AD), associated with AJ disorder, M5, PO, AG 5, NO, neuropathies (eg, neuropathic pain or alcoholic neuropathy), alcoholism or alcohol withdrawal, or spinal cord injury , in a subject in need thereof, comprising at least torasemide, trimetazidine, mesquiletin, ifenprodil, bromocriptine or moxifloxacin, or a salt(s), prodrug, derivative(s) or composition thereof) for sustained release, preferably in combination, with those described above.

In another embodiment, the present invention relates to a method of treating Alzheimer's disease (AD), a disorder associated with AI, M5, RO, AG5, NO, neuropathies (eg, neuropathic pain or alcoholic neuropathy), alcoholism or alcohol withdrawal, or a lesion spinal cord, in a subject in need thereof, which involves the simultaneous, separate or sequential administration to the subject of at least one first compound selected from the group consisting of torasemide, trimetazidine, mexiletine, ifenprodil, bromocriptine and moxifloxacin, salts thereof , prodrugs, derivatives or any compositions, in combination with at least one second compound, which is different from the first mentioned compound, and which is selected from sulfisoxazole, methimazole, prilocaine, diphylline, quinacrine, carbenoxolone, acamprosate, aminocaproic acid, baclofen , cabergoline, diethylcarbamazine, cinacalcet, cinnarizine, eplerenone, fenoldopam, leflunomide, levosimendan, sulodexide, terbinafine, zonisamide, etomidate, phenformin, three metazidine, mexiletine, bromocriptine, ifenprodil, torasemide and moxifloxacin or their salts, prodrugs, derivatives or forms with prolonged release.

In an additional embodiment, the invention relates to a method of treating Alzheimer's disease associated with AJ disorder, M5, PO, AG 5, NO, neuropathies (for example, neuropathic pain or

of alcoholic neuropathy), alcoholism or alcohol withdrawal, or spinal cord injury, in a subject in need thereof, with at least one first compound selected from the group consisting of torasemide, trimetazidine, mexiletine, ifenprodil, bromocriptine and moxifloxacin, salts thereof , prodrugs, derivatives or any compositions, in combination with at least one second compound, which is different from the first mentioned compound, and which is selected from sulfisoxazole, methimazole, prilocaine, diphylline, quinacrine, carbenoxolone, acamprosate, aminocaproic acid, baclofen, cabergoline, diethylcarbamazine, cinacalcet, cinnarizine, eplerenone, fenoldopam, leflunomide, levosimendan, sulodexide, terbinafine, zonisamide, etomidate, phenformin, trimetazidine, mexiletine, bromocriptine, ifenprodil, torasemide and moxifloxacin or their salts, prodrugs, derivatives or any compositions.

As described in the examples, in addition to being effective in protecting neurons from glutamate toxicity, baclofen-torasemide therapy is also particularly effective in stimulating neuronal cell growth, even in the absence of any exposure to a toxic agent or condition.

Moreover, ip mimo, this combined therapy leads to improvement in the loss of nerve signal transmission after nerve damage. Therefore, the baclofen-torasemide combination represents a new and powerful therapy for the treatment of the neuropathies, nerve lesions, and spinal cord lesions defined above.

Accordingly, in a specific embodiment, the invention relates to a method of treating neuropathies, such as peripheral nerve lesions or spinal cord lesions, which involves administering to a subject in need thereof a composition that includes salts, prodrugs, derivatives, or any compositions of baclofen and torasemide.

In another specific embodiment, the invention relates to a method of treating neuropathies, e.g., hereditary neuropathies, such as CMT disease, which involves administering to a subject in need thereof a composition comprising salts, prodrugs, derivatives, or any compositions of baclofen and torasemide .

In a more specific embodiment, the invention relates to a method of treating CMT! or CMT2 which involves administering to a subject in need thereof a composition comprising salts, prodrugs, derivatives or any compositions of baclofen and torasemide.

A specific object of the invention is also a method of treating neuropathies, e.g. lesions of the peripheral nerves 60, or lesions of the spinal cord, which involves the simultaneous, separate or sequential and/or repeated administration to a subject in need of such treatment of an effective amount of baclofen and torasemide as described above.

Despite the excellent efficacy of ip myo and ip mimo, depending on the subject or the specific condition, the methods and compositions of the present invention may be used in additional combination with additional drugs or treatments suitable for the treatment of a neurological condition in the subject. In this regard, in a specific embodiment, the drug(s) or compositions proposed by the present invention may be additionally combined with Sipkdo bBiora extracts. Suitable extracts include, without limitation, Sipkdo Bioba extracts, improved Sipkdo Bioba extracts (eg, enriched with active ingredients or in which the content of impurities is reduced), or any medicinal product containing Sipkdo Bibiora extracts.

Sipkdo Bioba extracts can be used in a composition that includes at least torasemide, trimetazidine, mexiletine, bromocriptine, ifenprodil and moxifloxacin.

In the best embodiments, Sipkdo Bioba extracts are used in combination with each of the following drug combinations: - acamprosate and ifenprodil; - baclofen and mexiletine; - baclofen and torasemide; - baclofen and trimetazidine; - bromocriptine and sulfisoxazole; - cinacalcet and mexiletine; - cinnarizine and trimetazidine; - eplerenone and torasemide; - sulfisoxazole and torasemide; - trimetazidine and zonisamide; - levosimendan and mexiletine; - levosimendan and ifenprodil; - levosimendan and trimetazidine; - levosimendan and moxifloxacin;

Zo - terbinafine and torasemide; - moxifloxacin and baclofen; - moxifloxacin and cinacalcet; - moxifloxacin and zonisamide; - moxifloxacin and sulfisoxazole; - mexiletine and ifenprodil; - baclofen and trimetazidine and torasemide; - baclofen and cinacalcet and torasemide; - baclofen and acamprosate and torasemide; - sulfisoxazole and trimetazidine and torasemide and zonisamide; - sulfisoxazole and mexiletine and torasemide and cinacalcet; - baclofen and acamprosate and torasemide and diethylcarbamazine; - baclofen and acamprosate and torasemide and diethylcarbamazine and ifenprodil; - levosimendan and baclofen and trimetazidine; - levosimendan and aminocaproic acid and trimetazidine; - levosimendan and terbinafine and trimetazidine, or - levosimendan and sulfisoxazole and trimetazidine.

Other therapies used in combination with the drug(s) or drug combination(s) of the present invention may include one or more drugs that alleviate the symptoms of Alzheimer's disease, AJ-related disorder, M5, PO, AI 5, NO, neuropathies (eg, neuropathic pain or alcoholic neuropathy), alcoholism or alcohol withdrawal, or spinal cord injury or drug(s), and which may be used for the palliative treatment of these disorders.

For example, the combinations proposed by this invention can be used in combination with donepezil (CA5: 120014-06-4), gabapentyl (SAB: 478296-72-9; 60142-96-3), galantamine (357-70-0), rivastigmine (123441-03-2) or memantine (SA: 19982-08-2).

Another object of the invention is the use of a compound or a combination of compounds described above for the manufacture of a medicinal product for the treatment of the above-mentioned disorders, by combined, separate or sequential administration of them to a subject in need thereof.

Another object of the invention is a method of obtaining a pharmaceutical composition, which involves mixing the above-mentioned compounds in a suitable filler or carrier.

The duration of treatment also depends on the stage of the disease or disorder being treated, the combination used, relapses and the condition of the patient, and how the patient responds to treatment. The dosage, frequency and route of administration of each component of the combination can be independently controlled. For example, one drug can be administered orally, while a second drug can be administered intramuscularly. Combination therapy can be given in intermittent cycles that include periods of rest so that the patient's body has a chance to recover from the unanticipated side effects that are still present.

Medicines can also be compounded so that a single injection delivers all the medicines.

The administration of each drug of the combination may be by any suitable means which produces a concentration of the drug which, in combination with the other component, is capable of ameliorating the condition of the patient or the effectiveness of the treatment of the disease or disorder.

While for the active ingredients it is possible to introduce the combination in the form of a pure chemical substance, it is preferable to present them in the form of a pharmaceutical composition, which is also referred to in this context as a pharmaceutical composition. Possible compositions include those suitable for oral, rectal, topical (including transdermal, buccal, and sublingual), or parenteral (including sublingual, intramuscular, intravenous, and intradermal) administration.

More often, these pharmaceutical compositions are prescribed to the subject in "patient packages" that contain dosage units or other means for administering measured unit doses for use during a separate period of treatment in a unit package, usually in a blister pack. Patient packs have advantages over traditional prescriptions, where the pharmacist separates the provision of the pharmaceutical to the patient from the bulk product, in that the patient always has access to the leaflet in the patient pack, which is usually absent from traditional prescriptions. Having a tab has been shown to improve compliance with doctor's instructions. Thus, the invention further includes a pharmaceutical composition, as previously described herein, in combination with a packaging material suitable for said compositions. In such packaging for the patient, the target application of the composition for combined treatment can be deduced by

With the help of instructions, devices, prescriptions, adaptations and/or other aids regarding the use of the composition, in the best way for the treatment. Such measures make the packaging for the patient particularly suitable and adapted for use in treatment with the composition proposed by the present invention.

The drug may be contained, in any desired amount, in any suitable carrier substance (eg, excipients, carrier, base), which may be 1-99% by weight of the total weight of the composition. The composition can be presented in a dosage form suitable for oral, parenteral (eg, intravenous, intramuscular), rectal subcutaneous, nasal, vaginal, inhalation, skin (patch) or ocular routes of administration. Thus, the composition may be in the form of, for example, tablets, capsules, pills, powders, granules, suspensions, emulsions, solutions, gels, including hydrogels, pastes, ointments, creams, patches, oral dispensers, osmotic delivery devices, suppositories, enemas, dosage forms for injections, implants, sprays and aerosols.

Pharmaceutical compositions can be formulated according to standard pharmaceutical practice (see, for example, Ketipdiop: Tpe 5siepse apa Rgasiise oi Rpagtasu (201st ea.), ey. A. Kk.

Seppago, I irripsoiyi M/Piatve 8 UMiIKip5, 2000 and Epsusioredia oi Rpaptaseciisa! Thesppoiodu, edv. "). zmaggisk apa u). S. Vouiap, 1988-1999, Magse! OekKeg, Mem/ MogK).

The pharmaceutical compositions of the invention can be formulated to release the active drug virtually immediately after administration or at any given time or period of time after administration.

Compositions for controlled release include (i) compositions that create a virtually constant concentration of the drug in the body for a long period of time; (ii) compositions that, after a predetermined lag period, create a virtually constant concentration of the medicinal product in the body for a long period of time; (its) compositions that maintain the action of the medicinal product for a predetermined period of time by maintaining a relatively constant effective level of the medicinal product in the body with the accompanying minimization of unwanted side effects associated with fluctuations in the plasma level of the substance of the active medicinal product; (im) compositions that localize the action of the medicinal product, for example, spatial arrangement of the composition with controlled release near or in the diseased tissue or organ; and (m) compositions that direct the action 60 of the drug using carriers or chemical derivatives to deliver the drug to a specific type of target cells.

Administering drugs in the form of a controlled-release formulation is particularly desirable in cases where the drug has a narrow therapeutic range (ie, a small difference between the plasma concentration that results in harmful side effects or toxic reactions and the plasma concentration that provides a therapeutic effect; in general, the therapeutic range, TD, is defined as the ratio of the half-lethal dose (O50) to the average effective dose (EU5o)); (ii) a narrow window of absorption in the gastrointestinal tract; or (ii) a very short half-life such that frequent dosing throughout the day is required to maintain therapeutic plasma levels.

Any number of strategies can be used to achieve a controlled release in which the rate of release exceeds the rate of metabolism of the drug in question. Controlled release can be obtained by appropriate selection of various composition parameters and ingredients, including, for example, different types of compositions and coatings for controlled release. Thus, the drug is formulated with appropriate fillers into a pharmaceutical composition, which, when administered, releases the drug in a controlled manner (single or multi-element tablets or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, nanoparticles, films and liposomes) .

Solid dosage forms for oral use

Compositions for oral use include tablets that contain the active ingredients in admixture with non-toxic pharmaceutically acceptable excipients. These excipients may include, for example, inert diluents or fillers (eg, sucrose, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (for example, cellulose derivatives, including microcrystalline cellulose, starches, including potato starch, croscarmellose sodium, alginates or alginic acid); binding agents (eg, acacia gum, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethylcellulose, methylcellulose,

From hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (eg, stearic acid, silicon oxide, or talc). Other pharmaceutically acceptable excipients may include coloring agents, flavoring agents, plasticizers, humectants, buffering agents, and the like.

The tablets may be uncoated or they may be coated by known methods, optionally to delay dissolution and absorption in the gastrointestinal tract, which provides delayed action over a longer period. The coating can be adapted to release the active drug substance in a predetermined pattern (for example, to obtain a controlled release composition) or the coating can be adapted not to release the active drug substance before passing through the stomach (enteric coating). The coating can be a sugar coating, a film coating (for example, based on hydroxypropyl cellulose, methyl cellulose, methylhydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, acrylate copolymers, polyethylene glycols and/or polyvinylpyrrolidone), or an enteric coating (for example, based on a copolymer of methacrylic acid , cellulose acetate phthalate, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate, polyvinyl acetate phthalate, shellac and/or ethyl cellulose.A time delay material such as glyceryl monostearate or glyceryl distearate may be used.

Solid tablet compositions may include a coating adapted to protect the composition from undesirable chemical changes (eg, chemical degradation prior to release of the active drug substance). The coating may be applied to the solid dosage form as described in the encyclopedia of pharmaceutical technology.

Medicines in a pill can be mixed together, or they can be separated.

For example, the first drug is held inside the tablet, and the second drug is held outside the tablet, so that a substantial part of the second drug is released before the release of the first drug.

Oral compositions may also be presented in the form of chewable tablets, or in the form of hard gelatin capsules, where the active ingredient is mixed with an inert solid solvent (eg, potato starch, microcrystalline cellulose, calcium carbonate, calcium phosphate, or kaolin), or in the form of m which 60 gelatin capsules, where the active ingredient is mixed with water or oil medium,

for example, liquid paraffin or olive oil. Powders and granules can be prepared using the ingredients mentioned above for tablets and capsules in the usual way.

Controlled release compositions for oral use can, for example, be designed to release the active drug by controlling the dissolution and/or diffusion of the active drug.

Dissolution- or diffusion-controlled release can be achieved by suitable coating of a tablet, capsule, pellet or granular drug formulation, or by incorporating the drug into a suitable matrix. Controlled release coatings may include one or more of the coating agents mentioned above and/or, for example, shellac, beeswax, Siusoulach, castor wax, carnauba wax, stearyl alcohol, glycerin monostearate, glycerin distearate, glycerin palmitostearate, ethyl cellulose, polyacrylates , OI -lactic acid, cellulose acetate-butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate, methyl methacrylate, 2-hydroxy methacrylate, methacrylate hydrogels, 1,3-butylene glycol, ethylene glycol methacrylate and/or polyethylene glycols. As part of a controlled release matrix, the matrix material may include, for example, hydrogenated methyl cellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate - methyl methacrylate, polyvinyl chloride, polyethylene and/or halogenated hydrofluorocarbon.

A controlled-release composition containing one or more of the claimed combination drugs may also be in the form of a floating tablet or capsule (ie, a tablet or capsule that, when administered orally, floats on the surface of the gastric contents for a period of time). The composition of the floating tablet of the drug(s) can be obtained by granulation of a mixture of the drug(s) with fillers and 20-7595 wt./wt. hydrocolloids such as hydroxyethyl cellulose, hydroxypropyl cellulose, or hydroxypropyl methyl cellulose. The resulting granules can then be pressed into tablets. When in contact with gastric juice, the tablet forms a practically waterproof gel barrier around its surface. This gel barrier is involved in maintaining a density of less than one, thereby allowing the tablet to remain buoyant in the gastric juice.

Lipids for oral administration

Powders, dispersible powders or granules suitable for preparing an aqueous suspension by adding water are common dosage forms for oral administration.

A suspension formulation provides the active ingredient in admixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives.

Suitable suspending agents are, for example, sodium carboxymethylcellulose, methylcellulose, sodium alginate, and the like.

Parenteral compositions

The pharmaceutical composition may also be administered parenterally by injection, infusion, or implantation (intravenous, intramuscular, subcutaneous, etc.) in dosage forms, compositions, or via suitable delivery vehicles or implants that contain conventional, non-toxic pharmaceutically acceptable carriers or adjuvants . Formulation and preparation of such compositions is well known to specialists in the field of pharmaceutical compositions.

Compositions for parenteral use may be presented in unit dosage forms (eg, in single-dose ampoules) or in vials that contain multiple doses and to which a suitable preservative may be added (see below). The composition may be in the form of a solution, suspension, emulsion, infusion agent, or delivery agent for implantation, or may be presented as a dry powder reconstituted with water or other suitable carrier before use. In addition to the active drug(s), the composition may include suitable parenterally acceptable carriers and/or excipients. The active drug(s) can be incorporated into microspheres, microcapsules, nanoparticles, liposomes, etc. for controlled release. The composition may include suspending, solubilizing, stabilizing, pH-adjusting agents and/or dispersing agents.

Pharmaceutical compositions proposed by the invention can have a form suitable for sterile injection. To prepare such a composition, a suitable active drug(s) is dissolved or suspended in a parenterally acceptable liquid carrier. Among the acceptable carriers and solvents that may be used are water, water adjusted to a suitable pH by the addition of an appropriate amount of hydrochloric acid, sodium hydroxide, or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution. The aqueous composition may also contain one or more preservatives (eg, methyl, ethyl or p-propyl p-60 hydroxybenzoate). In cases where one of the drugs is only slightly or poorly dissolved in water, a dissolution enhancing agent or solubilizing agent may be added, or the solvent may include 10-60 95 propylene glycol, etc.

Controlled-release parenteral compositions can be in the form of aqueous suspensions, microspheres, microcapsules, magnetic microspheres, oil solutions, oil suspensions, or emulsions. Alternatively, the active drug(s) may be incorporated into biocompatible carriers, liposomes, nanoparticles, implants or infusions. Materials for use in the manufacture of microspheres and/or microcapsules are, for example, capable of biological degradation /degradable polymers such as polyglactin, poly-(isobutyl cyanoacrylate), poly(2-hydroxyethyl-i-glutamine). Biocompatible carriers that can be used in the formulation of controlled-release parenteral formulations are carbohydrates (eg, dextrans), proteins ( e.g., albumin), lipoproteins, or antibodies Implant materials may be non-biodegradable (e.g., polydimethylsiloxane) or biodegradable (e.g., poly(caprolactone), poly(glycolic acid), or poly(ortho ethers) ).

Alternative ways

Other routes of introduction and therefore other compositions can be considered, although they are worse and less convenient. In this regard, suitable dosage forms of the composition for rectal use include suppositories (such as emulsions or suspensions) and rectal gelatin capsules (solutions and suspensions. In a typical suppository, the active drug(s) are combined with a suitable pharmaceutically acceptable suppository base, such as cocoa butter, esterified fatty acids, glyceryl gelatin and various water-soluble or dispersible bases such as polyethylene glycols.Various additives, enhancers or surfactants may be incorporated.

Pharmaceutical compositions may also be administered topically to the skin for transdermal absorption in dosage forms or compositions that typically contain non-toxic pharmaceutically acceptable carriers and excipients, including microspheres and liposomes.

Compositions include creams, ointments, lotions, liniments, gels, hydrogels, solutions, suspensions, sticks, sprays, pastes, patches and other types of transdermal drug delivery systems. Pharmaceutically acceptable carriers or excipients may include emulsifying agents, antioxidants, buffering agents, preservatives, humectants, penetration enhancers, chelating agents, gelling agents, ointment bases, perfumes, and skin protection agents.

Preservatives, moisturizers, penetration enhancers can be parabens such as methyl or propyl p-hydroxybenzoate and benzalkonium chloride, glycerin, propylene glycol, urea, etc.

The pharmaceutical compositions described above for topical administration to the skin may also be used in combination with topical administration on or near the part of the body to be treated. The compositions may be adapted for direct application or for application with special drug delivery devices such as bandages or otherwise plasters, pads, sponges, tapes or other forms of suitable flexible material.

Dosage and duration of treatment

It should be borne in mind that the drugs of the combination can be administered simultaneously, or one at a time, or in different pharmaceutical compositions, or sequentially. If sequential administration is used, the delay in the administration of the next (or additional) active ingredient should not be such as to lose the advantage of the active effect of the combination of active ingredients. The minimum requirement for a combination according to this description is that the combination should be intended for combined use with the advantage of the active effect of the combination of active ingredients. The intended use of the combination may be established by means of adaptations, prescriptions, adaptations and/or other means to aid in the use of the combination according to the invention.

Although the active drugs of the present invention can be administered in fractional doses, for example, two or three times a day, a single daily dose of each drug in combination is more preferred, with a single daily dose for all drugs in a single pharmaceutical composition (single dose form), which is most desirable. ee The term "unit dosage form" refers to physically discrete units (such as capsules, tablets, or prefilled syringe cylinders) suitable as unit dosages for human subjects, each unit containing a predetermined amount of active material or materials calculated to obtaining the desired therapeutic effect, in combination with the necessary pharmaceutical carrier.

Introduction, as a rule, is repeated. The administration can be carried out from one to several times daily for a period of time from several days to several years, and can even be carried out throughout the life of the patient. Chronic or, at least, periodically repeated administration is prescribed in most cases.

In addition, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic agent) information about a particular patient may influence the dosage used.

Except in response to specific cases of deterioration, where higher dosages may be required, preferably the dosage of each drug in the combination will be within the dosage range no higher than that normally prescribed for long-term maintenance treatment or for which safety has been demonstrated in phase C clinical trials. of research

One significant advantage of the invention is that each compound can be used in low dosages in combination therapy, while creating, in combination, a significant clinical benefit for the patient. Combination therapy can indeed be effective at doses where the compounds alone have virtually no effect. Accordingly, a specific advantage of the invention is the possibility of using each of the compounds in suboptimal doses, that is, doses that ARE lower than the therapeutic doses that are usually prescribed, preferably 1/2 of the therapeutic doses, preferably 1/3, 1/4, 1/5, or even better 1/10 of therapeutic doses. In specific examples, doses as low as 1/20, 1/30, 1/50, 1/100 or even lower than therapeutic doses are used.

At such suboptimal doses, the compounds individually will be essentially inactive, although the combination(s) proposed by the invention is fully effective.

The best dosages correspond to amounts from 1 956 to as much as 50 95 of those usually prescribed for long-term maintenance treatment.

The best dosage may correspond to amounts from 1 95 up to 10 95 of those usually prescribed for long-term maintenance treatment.

Specific examples of drug dosages for use in the invention are presented below: bromocriptine orally from about 0.01 to 10 mg per day, preferably less than 5 mg per day, more preferably less than 2.5 mg per day, even more preferably less than 1 mg per day, such dosages are available

Ko) especially suitable for oral administration; - ifenprodil orally from about 0.4 to 6 mg per day, preferably less than 3 mg per day, more preferably less than 1.5 mg per day, more preferably less than 0.75 mg per day, such dosages being particularly suitable for oral administration ; mexiletine orally from about 6 to 120 mg per day, preferably less than 60 mg per day, more preferably less than 30 mg per day, more preferably less than 15 mg per day, such dosages being particularly suitable for oral administration; moxifloxacin orally from about 4 to 40 mg per day, preferably less than 20 mg per day, preferably less than 10 mg per day, even more preferably less than 5 mg per day, such dosages being particularly suitable for oral administration; - torasemide orally from about 0.05 to 4 mg per day, preferably less than 2 mg per day, more preferably less than 1 mg per day, more preferably less than 0.5 mg per day, such dosages being particularly suitable for oral introduction; trimetazidine orally from about 0.4 to 6 mg per day, preferably less than 3 mg per day, more preferably less than 1.5 mg per day, more preferably less than 0.75 mg per day, such dosages are particularly suitable for oral administration; - acamprosate orally from about 1 to 400 mg per day; - aminocaproic acid orally from about 0.1 g to 2.4 g per day; - baclofen 0.01 to 150 mg per day, preferably less than 100 mg per day, more preferably less than mg per day, most preferably 5 to 40 mg per day, more preferably less than 35 mg per day, usually 15 50 mg per day, 12 mg per day, 24 mg per day, 30 mg per day, such dosages are particularly suitable for oral administration; - diethylcarbamazine orally from about 0.6 to 600 mg per day; - cinacalcet orally from about 0.3 to 36 mg per day; - cinnarizine from about 0.6 to 23 mg per day; - eplerenone orally from about 0.25 to 10 mg per day; - leflunomide orally from about 0.1 to 10 mg per day; - levosimendan orally from about 0.04 to 0.8 mg per day; - sulfisoxazole orally from about 20 to 800 mg per day; - oral sulodexide from about 0.05 to 40 mg per day; 60 - terbinafine orally from about 2.5 to 25 mg per day;

- zonisamide orally from about 0.5 to 50 mg per day.

It should be understood that the amount of drug actually administered will be determined by the physician, taking into account the attendant circumstances, including the conditions or circumstances of the subject being treated, the exact composition administered, the age, weight and response of the particular patient, the severity of the patient's symptoms, and the route chosen. introduction Therefore, the above dosage ranges are intended for general guidance and confirmation of what is stated in this document, but are not intended to limit the scope of the invention.

The following examples are presented for illustration purposes and are not intended to be limiting in any way.

Examples

Care and maintenance of animals, as well as experiments were carried out in accordance with the instructions

Committee on Research and Ethical Problems I.A.5.R. (1983).

A) Treatment of diseases associated with Ar toxicity

In these series of experiments, candidate compounds were tested for their ability to prevent or reduce the toxic effects of human AB:1-42. AVi-4»2 is a full-length peptide that is composed of aggregates found in biopsies of human patients affected by AI. Medicines were first tested separately, and then with tests for their combined effect. The effect was determined on different cell types, in order to further document the activity of the compounds in IP migo models that illustrate different physiological signs of AJ. In addition, research was also carried out in a mouse model to confirm the protective effect against AI, by evaluating the effect of compounds on i) cognitive activity of animals and i) molecular markers of AI (induction of apoptosis, induction of oxidative stress, induction of the inflammatory pathway).

I. Compounds that suppress the toxicity of human AV:-42

I.1. Protection against AB-42 toxicity in a model of human brain microvascular endothelial cells

Human brain microvascular endothelial cell cultures were used to investigate the protection afforded by the candidate compound(s) against AB'1-42 toxicity.

Microvascular endothelial cerebral cells of the human brain (NVMES, 5siepSei! Kegs. 1000, frozen at 10 passages) were rapidly thawed in a water bath at "37" C. The supernatant was immediately placed in 9U ml of Dulbecco's modified Eagle's medium (OMEM; Rap

From Viotesy gays: РО4-03600), which contains 10 96 fetal calf serum (EC5; SIVSO gays 10270-106). The cell suspension was centrifuged at 180 x d for 10 minutes at 4 °C and the sediment was suspended in C5C serum-free medium (C5C zegit ytee, Seiyi Zueyet, Keig ZE-4270-500-K, Batch 51407-4) with 1.6 96 "Zegit (Seyi Bubiet, Kei: 5E-420-500-K, Batch 54102), 2 to penicillin 10,000 Units/ml and streptomycin 10 mg/ml (RB5; Rap Bioesp gays: ROb-07100 batch 133080808) and seeded at a density of 20,000 cells per cell in 96-well plates (angiogenic system "Taigide! Iaueg Biosoai Apdiodepeuzi5 zuziet", VO, Kei 354150, Lot A8662) in a final volume of 100 μl. On the basis of matrigel, cerebral endothelial cells spontaneously started the process of capillary network morphogenesis (33).

Three separate cultures were obtained per condition, 6 cells per condition.

Treatment with candidate compounds and human amyloid-B:1-42

Briefly, peptide AVi--22 (Vaspet, gay H1IT368 Brisp 1010533) was reconstituted in a certain culture medium to 20 NM (stock solution) and slowly stirred at 37 °C for 3 days in the dark for aggregation. Control medium was obtained under the same conditions.

After 3 days, this aggregated human amyloid peptide was used on HBMES at a concentration of 2.5 µM, diluted in the control medium (optimal incubation time).

Peptide AB:-422 was added 2 hours after spreading НВМЕС on matrigel for 18 hours of incubation.

After one hour, HVMES was spread on matrigel, the test compounds and MES-165 were dissolved in the culture medium (0.1 95 OM5O) and then pre-incubated with HVMES for one hour before using AB:-42 (in the final volume of culture cell 100 μl). After an hour of incubation with the test compounds or MESE (two hours after seeding the cells on matrigel), 100 μl of AB:-42 peptide was added to a final concentration of 2.5 μM, diluted in control medium in the presence of test compounds or MESE (in a total volume of 200 μl/cell), in order to avoid further dilutions of the medicinal product.

Organization of cultural dies

MESE-165, known as the pro-angiogenic isoform of MESE-A, was used for all experiments in this study as a reference compound. MESE-165 is one of the most common isoforms of MESE involved in angiogenesis. MESE was used as a source of test compound at 10 nm. for The following states were evaluated:

- Negative control: medium alone with 0.1 96 DMSO - Intoxication: amyloid-B1-42 (2.5 NM) for 18 hours. - Positive control: MESE-165 (10 nM) (1 reference compound/culture) 1 hour before the addition of AB1-42 (2.5 NM) during the 18 h incubation period. - Test compounds: Test compounds 1 hour before adding AB1-42 (2.5 NM) for 18 hours. incubation period.

Quantitative analysis of the capillary network

2 images were taken per cell using IPSEI! Apaiuleg TM 1000 (SE Neaisage) in light transmission mode with 4x magnification. All images were obtained under the same conditions.

Analysis of angiogenesis networks was carried out with the help of RemeiIoreg software (SE

Neavsage). The total length of the capillary network was estimated.

Data Processing

All values are expressed as the mean (5.e. mean of cultures (n - 6 per condition). Statistical analyzes were performed under other conditions (by performing analysis of variance, then the criterion

Dannet, when possible, program (5iaimem zoimage megsiop 5.0). Values (as 9o) were inserted into graphs showing the evolution of amyloid toxicity. In fact, amyloid toxicity was taken as 100 95, and exposure to test substances was calculated as 95 of this amyloid toxicity.

The results

The results are presented in Figure 2. They demonstrated that the drugs individually induce a significant protective effect against the toxicity caused by the AB'-22 peptide: - Torasemide, in a low dosage, for example, 400 nM, induces a strong protective effect; - Bromocriptine, in a low dosage, for example, 3.2 NM, induces a strong protective effect.

The results also show that, unexpectedly, higher or lower drug concentrations compared to the above-mentioned drug concentrations may impair or have less or no effect on AB-42 toxicity in this model.

I.2 Protection against AB':-42 toxicity on primary cortical neuronal cells

Treatment with the test substance and human amyloid B1-42

Rat cortical neurons were cultured as described in Fig. (42). Briefly, pregnant female rats with 3 15-day pregnancies were killed by displacement of the cervical vertebrae (Kai5 Umiziag) and

Fetuses were removed from the uterus. The cortex was removed and placed in ice-cold Leibovitz medium (I 15) containing 2 95 penicillin 10,000 U/ml and streptomycin 10 mg/ml and 195 bovine serum albumin (B5A). The cortex was dissociated with trypsin for 20 minutes at 37 "C (0.05 95). The reaction was stopped by adding Dulbecco's modified Eagle's medium (OMEM), which contains DNase

Category II and 10 95 fetal calf serum (E5C). Cells were then mechanically dissociated with serial passages through a 10 ml pipette and centrifuged at 515 x r.p.d. for 10 minutes at -4 70. The supernatant was discarded and the cell pellet was resuspended in a specific culture medium containing Meygoras, enriched in B27 (2 95), I-glutamine (0.2 mM), 295 RZ solution and 10 ng/ml VOME. Live cells were counted in a Meiraceg cytometer using the trypan blue exclusion test. Cells were seeded at a density of 30,000 cells/cell in 96-well plates (cells were pre-coated with poly-I-lysine (10 μg/ml)) and cultured at 37 °C in humidified air (95 903/CO (5 95) atmosphere

Briefly, AB:-42 peptide was reconstituted in a specific culture medium at a concentration of 40 μM (stock solution) and slowly shaken at 37 °C for 3 days in the dark for aggregation. Control medium was obtained under the same conditions.

After 3 days, the solution was used on primary cortical neurons as follows:

After 10 days of culturing neurons, the drug was dissolved in the culture medium (40.195 DMSO) and then pre-incubated with neurons for 1 hour before the application of AB:-"2 (in a final volume per culture cell of 100 μl). After one hour of incubation of the drug(s), 100 μl of peptide AVD:1-422 was added at a final concentration of 100 μm diluted in the presence of the drug(s), in order to avoid further dilution of the drugs. Cortical neurons were intoxicated for 24 hours. Three separate cultures were obtained per condition, 6 cells per condition.

VOME (50 ng/ml) and estradiol-B (150 nM) were used as positive control and reference compounds, respectively.

Organization of cultural dies

Estradiol-VD at a concentration of 150 nm was used as a positive control.

Estradiol-B was dissolved in the culture medium and pre-incubated for 1 hour for use with aggregated amyloid B.-42.

The following conditions were evaluated: 60 - CONTROL SPOT: 12 cells/condition

- Negative control: medium alone - 0.196 DMSO - Intoxication: amyloid - B1-42 (10 μM) for 24 hours - Reference compound: Estradiol (150 nM) 1 hour. - MEDICINE TABLET: 6 cells/condition - Negative control: medium separately « 0.190 DMSO - Intoxication: amyloid - B1-42 (10 µM) for 24 hours - Drug: drug -1 h. then amyloid B1-42 (10 μM) for 24 hours

Lactate dehydrogenase (OH) activity test

24 hours after intoxication, the supernatant was separated and analyzed using a cytotoxicity detection kit (ION, Kospe Arriei Zsiepse, accession number: 11644793001, lot: 11800300). This colorimetric assay for the quantification of cellular cytotoxicity is based on the measurement of lactate dehydrogenase activity released from the cytosol of dying cells into the supernatant.

Data Processing

All values are expressed as mean (5.6. mean of cultures (n--6 per condition). Statistical analyzes were performed under other conditions (analysis of variance, followed by Dunnett's test when possible, Ziaimiem program, version 5.0).

The results

The results obtained for individual selected drugs in the toxicity assays on primary cortical neuronal cells are presented in Figures 2, 26 and 27. They demonstrated that the drugs individually induced a significant protective effect against the toxicity caused by the AB'-42 peptide: - trimetazidine, in a low dosage, for example, 40 nM, induces a strong protective effect; - mexiletine, already in a dosage of 3.2 NM, induces a strong protective effect; - bromocriptine, already in a dosage of 40 nM, induces a strong protective effect; - ifenprodil, already in a dosage of 3.2 NM, induces a strong protective effect; - moxifloxacin, already in a dosage of 20 nM, induces a strong protective effect. - torasemide, in a dosage of 200 nM, induces a strong protective effect. - Homotaurine, in a dosage of 8 nM, induces a strong protective effect.

The obtained results also unexpectedly show that concentrations of drugs higher and lower than those previously mentioned can worsen or have less or no protective effect against AB:-42 toxicity on neuronal cells.

II. Combination therapies prevent human AB:-42 toxicity

IP.1 The effect of combined therapies on the toxicity of the human ADVi-42 peptide on human LVMES cells.

The effectiveness of the combination of drugs proposed by the invention was evaluated on human cells. The protocol used in these tests is the same as described in section I.1 above.

The results

All drug combinations tested were protective against the toxicity of the human АВД:-42 peptide in the НВМЕС model, as shown in Table C below and illustrated in

Figures 3-6 and Figures 13-14. The results clearly show that intoxication with aggregated human amyloid peptide (AVi-42 2.5 IU) is significantly inhibited by the combinations proposed by the invention, while at these concentrations, the drugs alone do not have a significant effect on intoxication under the experimental conditions described above.

Table C

NVMES, intoxicated AB:-42 ifenprodil

Shown as examples in figures 3-6, 13 and 14, the following combinations of drugs give particularly interesting protective effects against the toxicity of the human peptide AB:i-42 during intoxication of HBMES cells: - baclofen and torasemide; - sulfisoxazole and torasemide; - torasemide and eplerenone; - sulfisoxazole and bromocriptine; - terminafine and torasemide, or - cinacalcet and mexiletine.

IP.28B The effect of combined therapies on the toxicity of human peptide A:-422 on primary cortical cells

The effectiveness of combinations of drugs of the invention was evaluated on primary cortical neuronal cells. The protocol used in these tests is the same as described in section I.2 above.

The results

All drug combinations tested were effective against the toxicity of the human AVI1-42g2 peptide in primary cortical neuronal cells, as shown in Table 4 below and illustrated in Figures 7-12 and 16-22. The results clearly show that intoxication with aggregated human amyloid peptide (AVi-22 10 μm) is significantly inhibited by the combinations proposed by the invention, while at these concentrations, the drugs alone do not have a significant effect on intoxication under the experimental conditions described above.

Table 4 000 zhnantsnyayayunnyanyu Le

A combination of drugs AB:-4g primary cortical neuronal cells

Es ionni nny PI UNN ifenprodil x

Given as examples in Figures 7-12, 15-22, the following drug combinations give particularly interesting protective effects against the toxicity of the human peptide AB.-422 in cell intoxication

NVMES: - acamprosate and ifenprodil; - baclofen and mexiletine; - baclofen and torasemide; - baclofen and trimetazidine; - cinacalcet and mexiletine; - cinnarizine and trimetazidine; - trimetazidine and zonisamide; - mexiletine and ifenprodil; - moxifloxacin and baclofen; - moxifloxacin and cinacalcet; - moxifloxacin and trimetazidine; - moxifloxacin and sulfisoxazole; - moxifloxacin and zonisamide, or - torasemide and sulfisoxazole. 11. Protection of neurite growth from AB1-42 toxicity

Test compounds and processing AB':-42

Primary rat cortical neurons were cultured as previously described.

After 10 days in culture, the cells were incubated with drugs. After 1 hour, cells were intoxicated with 2.5 μM beta-amyloid (1-42; Vaspet) in a defined medium without VOME, but together with drugs. Cortical neurons were intoxicated for 24 hours.

VOME (10 ng/ml) was used as a positive (neuroprotective) control. Three separate cultures were obtained per condition, 6 cells per condition.

The length of the neurites

After 24 hours of intoxication, the supernatant was separated and cortical neurons were fixed with a cold solution of ethanol (95 95) and acetic acid (5 95) for 5 min. After permeabilization with 0.1 95 saponin, cells were blocked for 2 hours with PB5 containing 1 95 fetal calf serum. The cells were then incubated with a monoclonal antibody against 2 protein associated with microtubules (tisgolybcie-aziosiyed-rgoivip 2, MAR-2; 5idta).

This antibody was detected using a goat anti-mouse Ids antibody labeled Aihe EriIog 488 (MoiIesciag rgobe). Nuclei of neurons were labeled with a fluorescent marker (Noespvzi voYihop, 5ISMA).

10 pictures were taken on the cell with the help of the IPSEI analyzer! Apaiulegt"M 1000 (SE

Neapyrsage) with 20 x magnification. All images were obtained under the same conditions. The analysis of the neurite network was carried out using the YuemeIoreg software (ZE Neaysage) to estimate the total length of the neurite network.

The results

The combination of baclofen and torasemide induces a significant protective effect against the toxicity of human peptide AB:i-42 (an increase in neurite network by 531 95) in primary cortical neuronal cells, as shown in Figure 23. The results clearly show that intoxication with human amyloid peptide (AB:- 42 2.5 NM) is significantly inhibited by the combination, and that, however, the combination enhances the neurite network compared to the control.

Therefore, this combination allows you to effectively protect cortical neuronal cells and cells of the neuronal network against the toxicity of the human AVi-42 peptide. Moreover, this increase in the neurite network supports the efficacy of such drugs in neurological disorders such as spinal cord injury.

I. The compounds prevent the toxicity of human AVg5-z5 ip mimo

From the Animal

5m/iz5 male mice were used throughout the study. Animals were housed in plastic cages with free access to laboratory feed and water, except for behavioral experiments, and kept in a controlled environment, at 12 h. alternating cycles of light and darkness (the light was turned on at 8 am). Behavioral experiments were performed in a soundproof experimental room with controlled air circulation, to which mice were allowed to acclimate for at least 30 min before each experiment.

Production and injection of amyloid peptide

АВг»-з5 peptide and scrambled АВг5-з5 peptide were dissolved in sterile bidistilled water, they were stored at -20"С until use. Observation using light microscopy showed that incubation of АВг5-зх peptide, but not scrambled АВг5-з5 peptide Leads to the appearance of two types of insoluble precipitates, birefringent fibril-like structures, and amorphous spherical aggregates. B-amyloid peptides were then administered intracerebroventricularly (i.p.m.). Briefly, each mouse was lightly anesthetized with ether, a steel needle was inserted unilaterally 1 mm into the right from a midline point equidistant from each eye, equidistant between the eyes and ears, and perpendicular to the plane of the skull. Peptides or vehicle were delivered gradually over approximately 3 s. Mice displayed normal behavior within 1 min of injection. The injection site was checked injection of India ink in previous experiments.Neither needle insertion nor vehicle injection had a significant effect on the behavior, behavioral responses and cognitive functions.

Treatment with medicinal compound(s)

On day 1, that is, 24 hours before the injection of the АВг25-з5 peptide, drugs, combinations of drugs, or a carrier solution were administered orally by gavage twice a day (at 8 am and 6 pm).

On the 10th day (at 10 a.m.), mice were injected i.s.m. peptide АВг25-з5 or scrambled peptide АВгв-з5 (control) in a final volume of 3 μl (3 mM).

Between day 0 and day 7, drugs, combination drugs, or vehicle were administered orally by gavage twice daily (at 8 am and 2 pm). One animal of the group received donepezil (reference compound, 1 mg/kg/day) orally by gavage, one injection (at 8 am). Medicines were solubilized in water and prepared anew before each probe

Gs10) introduction.

On day 7, all animals were tested for the efficiency of spontaneous alternation in the test with the U-shaped maze, according to the indicator of spatial working memory.

On day 7 or 8, animals' contextual long-term memory was assessed using a step-down passive avoidance procedure.

On day 8, mice were killed. Their brains were dissected and stored at -80 C until further analysis.

Positive results were observed in behavioral activities and biochemical tests performed 7 days after i.s.m. injections of the ABg5-c5 peptide, namely for the combinations listed in Table 5.

Table 5

Biochemical results

A combination of drugs and/or behavioral tests ifenprodil

IM. The compounds enhance the behavioral and cognitive activities of intoxicated animals

Animals were intoxicated as indicated in the section above.

Effectiveness of spontaneous alternation - Test in a U-shaped maze.

On day 7, all animals were tested for spontaneous alternation performance in the U-maze, a measure of spontaneous working memory. The U-shaped maze is made of gray polyvinyl chloride. Each shoulder is 40 cm long, 13 cm high, and 3 cm wide at the bottom, 10 cm wide at the top, and the shoulders meet at a single point with an equal angle. Each mouse was placed at the end of one arm and allowed to move freely through the maze during an 8-minute session. Series of measures in the shoulder, including possible returns to the same shoulder, were checked visually. Alternation is defined as steps in all three shoulders in each subsequent step. The number of maximum alternations is therefore the total number of events in the arm, minus two, and the percentage of alternations is calculated as (actual alternations/maximum alternations) x 100. The parameters include the percentage of alternation (a measure of memory) and the total number of events in an arm (a measure of study ). Animals that demonstrated extreme behavior (percentage of alternation « 25 95 or » 85 95 or the number of steps in the "shoulder" of the maze « 10) were rejected. As a rule, rejection was 0-5 95 animals. This test, in particular, serves to analyze the contribution of the behavioral level and the amnesic effect induced in mice by injection of AVg5-z5.

Test of conditioned passive avoidance reflex

The apparatus is a two-chambered (15 x 20 x 15 cm high) box with one chamber illuminated by white PVC walls and the other chamber darkened by black PVC walls and a mesh floor. A guillotine door separates the cells. A 60-watt lamp located 40 cm above the equipment illuminates the light chamber during the experiment. Randomized electropain stimuli to the labia of the animals (0.3 mA for 3 seconds) can be performed on the mesh floor using a shock generator with a stirrer (Iagauetse Ypzigitepih, Lafayette, USA). The liftgate is initially closed during the training session. Each mouse was placed in a light chamber. After 5 seconds, the door rose. When a mouse entered the darkened chamber and placed all of its labia on the mesh floor, the door was closed and an electric shock was delivered for 3 seconds.

Step latency, i.e., the latency spent entering the darkened chamber, and the number of vocalizations were recorded. The retention test was performed after 24 hours. after training Each mouse was again placed in the light chamber. After 5 seconds, the door was raised, and the step delay and escape delay, that is, the time spent returning to the light chamber, were recorded up to 300 seconds.

Positive results were observed for each of the tested combinations listed in Table 6.

Table 6

Test in

The combination of drugs is like a maze of escape delay

M. The compounds proposed by the invention improve the neurophysiological understanding of neurological diseases

Combined therapies were tested in an IP model of AD intoxication. We evaluated their influence on some parameters that are disturbed in neurological diseases: - The level of expression of caspases C and 9, considered as an indicator of apoptosis; - Peroxidation of lipids, considered as a marker of the level of oxidative stress; - CEAR expression test, considered as a marker of the level of inflammation in the brain; - Integrity of the blood-brain barrier; - The overall integrity of synapses (tIFA for synaptophysin).

Integrity of the blood-brain barrier

The experimental design of animal intoxication with AB was the same as in part PI.

The potential protective effect of combined therapies on the integrity of the blood-brain barrier (BBB) was analyzed in mice injected intracerebroventricularly (i.s.m.) with oligomeric amyloid-B25-z5 peptide (АВг5-з5) or scrambled АВг5-з5 control peptide ( 5c.AB), 7 days after the injection.

On day 7 after the injection of AVg5-z5 animals, the integrity of the BBB was tested using the method with EV (Emap5 Vipe). EB dye is known to bind to serum albumin after peripheral injection and is used as a tracer of serum albumin.

Zo Barvnyk EV (295 in saline solution, 4 ml/kg) was injected intraperitoneally (i.v.) 3 hours before transcardial perfusion. Mice were anesthetized using v.o. 200 μl of a preliminary mixture of ketamine 80 mg/kg, xylazine 10 mg/kg, opened the chest.

Mice were transcardially perfused with 250 ml of saline for about 15 minutes. until the fluid from the right atrium became colorless. After decapitation, the brain was removed and subjected to dissection in three areas: cerebral cortex (left and right), hypothalamus (left and right), diencephalon. Each brain region was then weighed to quantify EB-albumin extravasation.

Samples were homogenized in phosphate-buffered saline and vortexed after addition of 60 95 trichloroacetic acid to precipitate protein. The samples were cooled at 4 "C, then centrifuged for 30 minutes at 10,000 49.4 "C. The supernatant was measured at 610 nm for EV absorption using a spectrophotometer.

EB was quantified and as - μg/kg of brain tissue using a standard curve obtained using a known concentration of EB-albumin. - μg/kg of protein.

As mentioned in Table 7, the combined therapies proposed by the invention are effective in maintaining the integrity of the BBB compared to untreated intoxicated animals.

General integrity of synapses (tIFA for synaptophysin)

Synaptophysin was chosen as a marker of synapse integrity and tested using a commercial tIFA kit (Sh5SM, Kei. E90425My). Samples were obtained from hypothalamic tissues and homogenized in extraction buffer as described by the manufacturer and in the relevant literature.

Tissues were washed with ice-cold PB5 (0.02 mol/l, pH 7.0-7.2) to completely remove excess blood and weighed before freezing in nitrogen and storing at -80 °C. Tissues were cut into small pieces and homogenized in 1 ml ice-cold phosphate-buffered saline (PB5) using a glass homogenizer. The resulting suspension was sonicated with an ultrasonic cell disintegrator during two freeze-thaw cycles to further disrupt cell membranes. The homogenates were then centrifuged for 5 minutes at 5000 49 and the supernatant was evaluated immediately.

All samples were tested in triplicate.

Quantification of proteins was carried out using a kit for determining the amount of protein with

BCA (bicinchoninic acid) by Riegse (Riegse, Kei. 2423227) to evaluate the efficiency of extraction and carry out normalization.

Total protein concentrations were then calculated from standard curve dilutions and used to normalize tIFA results.

The results (Table 7) demonstrate that the combined therapies are effective in maintaining the total level of synaptophysin in the brain of treated animals, compared to untreated intoxicated animals.

Oxidative stress test

Mice were killed by decapitation and both hippocampi were quickly removed, weighed, and stored in liquid nitrogen until testing. After thawing, hypothalamus was homogenized in cold methanol (1/10 wt./o6), centrifuged at 1000 9 for 5 minutes, and the supernatant was placed in an Eppendorf tube. The reaction volume of each homogenate was added to 1 mm Eeso4, 0.25 M

Na5O", 1 mm xylenol yellow-hot and incubated for 30 minutes at room temperature. After reading at 580 nm (A580 1), 10 μl of 1 mM cumene hydroperoxide (CHP) was added to the sample and incubated for 30 minutes at room temperature to determine the maximum level of oxidation. Absorption was measured at a wavelength of 540 nm. The level of lipid peroxidation is determined as SNR equivalents (SNR) according to: SNR - A58O 1/A580 2 x |SNR) and was expressed as SNR equivalents per tissue mass and as a percentage of the data of the control group.

The results (Table 7) show that combined therapies are effective in reducing

Of the total oxidative stress induced by AB in the brain of treated animals compared to untreated intoxicated animals.

Caspase pathway induction test and OBAR expression test

Mice were killed by decapitation and both hippocampi were quickly removed and washed in ice

RVZ (0.02 mol/l, pH 7.0-7.2) to completely remove excess blood, was weighed and stored in liquid nitrogen until testing. Tissues were cut into small pieces and homogenized in 1 ml of ice-cold PB5 using a glass homogenizer. The resulting suspension was sonicated using an ultrasonic cell disintegrator during two freeze-thaw cycles for further destruction of cell membranes. The homogenates were then centrifuged at 5000 g for 5 minutes and the supernatant was evaluated immediately.

Experiments were performed using a commercial Caspase-3 test (Ш5СМ-Е90626Мы),

Caspase-9 (05SM-E90627My), SEAR (O05SM-E90068).

Quantification of proteins was carried out using a kit for determining the amount of protein with

BCA (bicinchoninic acid) by Riegse (Riegse, Kei. 223227) to evaluate the efficiency of extraction and carry out normalization.

The results (Table 7) demonstrate that the combined therapies affect markers of apoptosis and inflammation in the brains of treated animals, compared to untreated intoxicated animals.

Table 7

Combination. Oxidative Expression Integrity Total Drugs Caspase Pathway Stress SEAR BBB Synapse Integrity

TORASEMIDE baclofen- acamprosate- sh sh sh sh sh sh torasemide

В-- "NNI NNNNEZNNN NINEUNNI NNYA PENY SHOE nya nya nya nya nya price

E---nshiy NNISHEZNNY NINEZNNY NIZYN PENI NOENOYA and nyan nyan nyan nyan torasemide

C) prevention of glutamate toxicity on neurons

In this additional series of experiments, candidate compounds were tested for their ability to prevent or reduce the toxic effects of glutamate toxicity in non-neuronal cells.

Glutamate toxicity has been implicated in the pathogenesis of neurological diseases or disorders such as multiple sclerosis, Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, neuropathies, alcoholism or alcohol withdrawal, or spinal cord injury. Medicines were first tested individually, and then tests were conducted on their combined effect.

Methods

The effectiveness of combinations of drugs proposed by the invention was evaluated on primary cortical neuronal cells. The protocol used in these tests is the same as described in section A.I.2 above.

Glutamate toxicity tests

The neuroprotective effect of the compounds was assessed by quantification of the neurite network (neurofilament (ME) staining), which specifically detects glutamatergic neurons.

After 12 days of neuron culture, drugs of the candidate combinations were dissolved in the culture medium (10.1 95 DMSO). Candidate combinations were then pre-incubated with neurons for 1 hour prior to glutamate challenge. After one hour of incubation, glutamate was added for 20 minutes, to a final concentration of 40 μM, in the presence of the candidate combinations, in order to avoid further dilution of the drugs. At the end of the incubation, the medium was replaced with medium containing the candidate combination but without glutamate.

The culture was fixed 24 hours after glutamate challenge. MK8O01 (disocycline maleate, 77086-22-7 - 20uM) was used as a positive control.

After permeabilization with saponin (Zidta), cells were blocked for 2 h with

PB5 containing 10 95 goat serum, then the cells were incubated with mouse monoclonal primary antibody against neurofilament antibody (ME, Zidt). This antibody was detected using a goat anti-mouse Ids antibody conjugated to AiIe

Ninog 488.

The nuclei of cells were labeled with a fluorescent marker (Noespv5i zoYshiop, ZIZMA), and the neurite network was quantified. Six cells per condition were used to assess neuronal survival in different cultures.

The results

All tested drug combinations provide a protective effect against glutamate toxicity to cortical neuronal cells. The results are shown in Table 8 below.

As illustrated in Figures 24 and 25, the combinations proposed by the invention better protect neurons from glutamate toxicity under the experimental conditions described above. Notably, effective protection is reported using drug concentrations at which the drugs used alone had no significant or low protective effect.

Table 8 glutamate

C) improvement in other disorders associated with glutamate excitotoxicity using combinations of the invention

The aforementioned protective effect against glutamate toxicity of the drugs and combinations proposed by the invention, in combination with the protective effects illustrated herein in several models of AO, prompted the inventors to test these drugs in some models of other diseases in the pathology of which glutamate toxicity is also involved, such as M5, AI 5 and neuropathic pain.

I) Protective effect of combinations in IP mimo model of multiple sclerosis

The model in which chronic progressive EAE develops in myelin-oligodendrocyte glycoprotein-immunized (MOb-immunized) mice was used to demonstrate the beneficial effect of the compositions proposed by the invention in the treatment of multiple sclerosis.

Animals and chemicals

C571/62 female mice (8 weeks old) were purchased from dapmieg (France); after two weeks of adaptation, female mice (10 weeks old) develop chronic paralysis after immunization with MO peptide (myelin oligodendrocyte glycoprotein). Experimental encephalomyelitis is induced using the NookKe Kii MOSz5-55/CEA Etiviop RTX (pertussis toxin) kit for EAE induction (EK-0110, EK-0115; NookKe Iabogafogie5). The control set is SK-0115 (NooKe

Iabotayughiev5).

Experiment procedure

Experimental encephalomyelitis was induced by the following procedure:

On day 0, two subcutaneous injections of 0.1 ml each were made; one to the upper back of the mouse, and the other to the lower back. Each injection contains 100 μg peptide МОсз5-55 (MEMOM/UKZREBAKUMNI UKMOK), 200 μg of inactivated Musobasiegisht yibegsshtsov5ziz NZ7Ka emulsified in Freund's complete adjuvant (CEA) (NooKe Iabogayogie5). The emulsion provides the antigen necessary for the spread and differentiation of MOC-specific autoimmune T-

From the cells.

Two intraperitoneal injections of 500 ng of pertussis toxin in PB5 (Nooke kit) were performed 2 hours (day 0) and 24 hours (day 1) after the injection of MOSS. Pertussis toxin enhances the development of EAE by providing an additional adjuvant.

Mice develop EAE 8 days after immunization and remain chronically paralyzed throughout the experiment. After immunization, mice were observed daily for clinical signs in a blinded manner. Animals were housed in a routine pathogen-free technical unit, and all experiments were performed according to the guidelines described or approved by the official local bioethics committee.

Experimental groups and drug treatment:

Groups of female mice were homogenized by weight before immunization as described: - Control group: vehicle injection under the same conditions to mice with EAE (from day 1 to day 28, placebo was given daily). - EAE group: MOSS injection (day 0) "from pertussis toxin injection (day 0 and 1) - from day 1 to day 28, placebo is given orally every day. - EAE and positive control: injection of MOS (day 0) "injection of pertussis toxin (day 0 and 1) - from day -1 to day 28, dexamethasone was given orally every day. - EAE "5 treatment group: MOS injection (day 0) x pertussis toxin injection (day 0 and 1).

Treatments begin the day before immunization and end by day 28.

Clinical scores were measured on days 0-5-8-9-12-14-16-19-21-23-26-28.

Biabis software (Z(afoi ps.)) was used throughout for statistical analysis. Analysis of variance and Student's t test were used to analyze the clinical score of the disease. P < 0.05 was considered significant.

Zo

Delays to disease onset, clinical score, and delay in death were compared between each group with the reference "itta" group using Kaplan-Meier curves and the model

Coke (package A "survival"). The obtained p-values are one-sided and test the hypothesis of improvement compared to the reference "itta" group.

The total clinical score is composed of the tail score, the hindquarters score, the forequarters score, and the bladder score as described below:

Tail score:

Back limb score:

Point--1 Each of the following tests is positive:

A - Rollover test: holding the tail between the thumb and forefinger, turn the animal onto its back and record the time it takes for the animal to roll over. A healthy mouse will roll over immediately. Delay suggests weakness of hind limbs.

B - Place the mouse on the top grid of the cage and watch it cross it from one side to the other. If one or both limbs often slip between the bars, then it is considered that there is partial paralysis.

Ball-Z3 One or both hind limbs have signs of paralysis, but some movements are preserved; for example: an animal may grasp and hold onto the underside of the top mesh of a cage for a short period of time before being released.

Ball of the front limbs: keep their head up.

Score-1 Movement is possible but difficult due to weakness of one or both labia, for example, the anterior labia is considered weak when the mouse has difficulty gripping the underside of the upper mesh of the cage. Another sign of weakness is a lowering of the head.

Score-2 When one front limb is paralyzed (impossibility to grasp anything and the mouse turns around the paralyzed limb). At this time, the head loses most of its muscle tone.

The ball of the bladder is soaked with urine.

The total score for each animal was determined by adding all the above categories.

The maximum score for live animals was 10.

Results - combined therapies are effective in the M5 model.

A significant improvement in the global clinical score is observed in the mice of the treatment group

EAEn-", namely for the combinations listed in Table 9.

Table 9

A combination of medicines

EAE-animals 11. Protective effect of combinations in AI model 5

Combination therapies of the present invention have been tested ip migo, in a co-culture model, and ip mimo, in a mouse model of AH 5. Protocols and results are presented in this section.

I.1 Protective effect against glutamate toxicity in primary cultures of neuromuscular joint culture

Primary joint cultures of nerve and muscle cells

Human muscle was obtained according to a previously described method from part of biopsies from a healthy patient (44). Muscle cells were obtained from dissociated cells (10,000 cells per cell), plated on gelatin-coated 0.1 95 by 48-well plates and grown in a proliferation medium containing a mixture of MEM medium and M199 medium.

Immediately after the fusion of the eyes, it is necessary to achieve a good ratio of innervations.

Innervated cultures were maintained in mixed medium. After 24 hours, with the usual co-cultivation of neurites, the growth of spinal cord explants was observed. They can contact the muscular tubules and induce the first contractions after 8 days. Immediately after that, the innervated muscle fibers are located in the immediate vicinity of the explants of the spinal cord, and they contract almost continuously. Innervated fibers are morphologically spatially different from non-innervated fibers and can be easily distinguished from the rest.

One co-culture completed (6 cells per condition).

Glutamate damage

On day 27, co-cultures were incubated with candidate compounds or riluzole for one hour before intoxication with glutamate (60 μM) for 20 minutes. Co-cultures were then washed and candidate compounds or riluzole were added for an additional 48 hours. After this incubation time, unfixed co-cultures were incubated with α-bungarotoxin coupled to

AIekha 488, at a concentration of 500 nmol/l for 15 minutes at room temperature. Then, co-cultures were fixed by REA for 20 minutes at room temperature. After permeabilization with 0.1 95 saponin, cocultures were incubated with anti-neurofilament antibody

Co., Ltd. (ME).

These antibodies are detected using the AiYeha Risog 568 goat anti-mouse antibody

Icho (MoiIesshag rgobe). Nuclei of neurons are labeled with a fluorescent marker (Noespvi zoHyop).

Endpoints are (1) total neurite length, (2) number of motor units, (3) total motor unit area, which is indicative of motoneuron survival and functionality.

For each condition, 2 x 10 images were taken with the help of an Ipsei analyzer Apaiu7geg TM 1000 (SE Neasage) with 20 x magnification. All images were obtained under the same conditions.

The results

Medicines proposed by the invention effectively protect motoneurons and motor units in a co-cultivation model.

Table 10

Protective effect against intoxication

Combination of drugs with glutamate in muscle/nerve co-cultures

I.2 - Combined therapies are effective in the AI 5 mouse model

Experiments were performed on male mice. Male transgenic mice B65)- 19«5001)20y117) and their control (respectively, 5M2726 and 5M2297, from daskzop I aroghaigie5, Ben Harbor,

USA, distributed in France by Spagieh Kimeg) were chosen for this set of experiments to simulate AG 5.

Affected mice express the 5001-593A transgene, constructed from a mutant human 5001 gene (single amino acid substitution of glycine to alanine at codon 93) driven by its endogenous human 5001 promoter. Control mice express the control human 5001 gene.

Administration of the medicinal product

Mice were administered doses of drug candidates diluted in a carrier from the 60th day after birth until death. Diluted solutions of drug candidates were prepared with water at room temperature immediately before administration. - In drinking water:

Riluzole was added to drinking water at a final concentration of 6 mg/ml (adjusted to the average body weight of each group) in 5 95 cyclodextrin. Since the mice drank about 5 ml/day, the estimated dose administered is 30 mg/kg/day, which is the dose that has been shown to increase the survival of the mice. - Cyclodextrin was used as a carrier in a final concentration of 5 95, diluted with water at room temperature from the stock solution (cyclodextrin 20 95). - Oral administration (by mouth): - Drug combinations were administered orally daily. - Cyclodextrin was used as a carrier in a final concentration of 5,956, diluted with water at room temperature from the stock solution (cyclodextrin 20,965).

Clinical observation

Clinical observation for each mouse was performed daily from the first day of treatment (60 days of age) until death (or sacrifice). Clinical observation includes study of behavioral tests: onset of paralysis, "loss of extension", "loss of rollover reflex" and general observation of gait: - occurrence of paralysis: observation includes observation of paralysis in each limb.

From The occurrence of paralysis corresponds to the day of the first signs of paralysis. - The hind limb loss test involves recording tremors or shaking and the position of the hind limbs (sag or skew) when the mice are held by the tail. - In the loss of rollover reflex test, the ability of mice to roll over was assessed

30 seconds when turning to any side. The rollover reflex is lost when the mouse is unable to roll itself over. The loss of the rollover reflex is determined in the final stage of the disease: mice unable to roll over are euthanized.

Results - Combined therapies are effective in ip mimo model AI 5

Improvement of the disease is observed in sick animals treated with drugs and combinations of drugs proposed by the invention. It should be noted that the combinations of drugs listed in Table 11 effectively improve the clinical score of these animals during various stages of the disease.

Table 11

I) Protective effect of the combinations of the invention in oxaliplatin-induced neuropathy as an IP mimo model of neuropathic pain

The combined therapies proposed by the present invention have been tested ip mimo in a suitable model of peripheral neuropathy, i.e. an acute model of oxaliplatin-induced neuropathy and a chronic model of oxaliplatin-induced neuropathy. Animals, protocols, and results are presented in this section.

Keeping of animals

Sprague-Dawley rats (SEC, France) with a weight of 150-175 g at the beginning of the oxaliplatin (bo) treatment experiments were used. The animals were housed in a technical block for work with animals with limited access in a room with controlled temperature (19.5 "C - 24.57) Its relative humidity (19.5 "C - 24.5 C) with a 12-hour cycle of light/ dark, with ad libitum access to standard pelleted laboratory chow and water throughout the study.Animals were housed 4 or 5 per cage and observed for a week's acclimatization period before any testing.

Experiment plan:

The following four groups of rats were used in all experiments:

Control groups:

Group 1: Oxaliplatin carrier (distilled water), v.o. /Carrier of the candidate combination(s) (distilled water), p.o. daily.

Group 2: Oxaliplatin (distilled water), v.o. /carrier of the candidate combination(s) (distilled water), p.o. daily.

Group 3: Oxaliplatin C mg/kg i.o./single drug in distilled water, p.o. every day x 9.

Tested composition of the group:

Group 4: Oxaliplatin Z mg/kg i.o./candidate combination(s) in distilled water, p.o. every day x 9.

Group 5 6 oxaliplatin C mg/kg PO/gabapentin (100 mg/kg) in distilled water, orally on test days (ie O: and Ov);

Vehicle and test units were delivered daily from 0-1 to 07 (the day before the last day of testing), whereas gabapentin was administered on test days (120 minutes before the test).

All treatments were coded and randomized whenever possible. Doses are expressed according to the indicator of free active substance.

Induction of neuropathy

Acute neuropathy was induced by a single intraperitoneal injection of oxaliplatin (3 mg/kg).

Chronic peripheral neuropathy was induced by repeated intraperitoneal injections of oxaliplatin (3 mg/kg, i.o.) on days 0, 2, 4, and 7 (Cb0-12 mg/kg, i.o.). Chronic neuropathy in humans is cumulative and is also most common in patients who received total doses of oxaliplatin "or -540 mg/m", which correspond to "15 mg/kg as a cumulative dose in

From rats (Segzovzito K.). 2005).

Oxaliplatin-induced painful neuropathy in rats reproduces painful symptoms in oxaliplatin-treated patients: - Thermal hyperalgesia is the earliest symptom. It can be measured using the acetone test or the tail dip test; - Mechanical hyperalgesia appears later. It can be quantified using the von Frey test or the paw pressure test.

Dosing and animal testing

All drug combinations were administered from the day before the first intraperitoneal injection of oxaliplatin 3 mg/ml (0-1) and continued to be administered orally until 07. During the test days (ie, 01 and 07), the drug combinations were administered after the test. Animals from the group treated with the standard (gabapentin) were administered only on test days.

Acetone test

Cold allodynia was assessed using the acetone test by measuring responses to thermal non-painful stimulation at 01 (about 24 hours after the first injection of oxaliplatin 3 mg/kg (acute effect of oxaliplatin)) and 08 (chronic effect of oxaliplatin).

In the acetone test, the hindpaw withdrawal delay is measured after applying a drop of acetone to the plantar surface of both hindpaws (reaction time) and the intensity of the response is calculated (cold score). The reaction time to the cooling effect of acetone is measured within 20 seconds (cut-off time) after application of acetone. Responses to acetone are also graded on the following 4-point scale: 0 (no response); 1 (quick twitching, jerking of the labia); 2 (prolonged twitching or noticeable jerking of the labia); C (repeated jerking of the labia with licking or biting).

For each experimental group, the results are expressed as:

The reaction time is defined as the time, expressed in seconds, required to detect the reaction of the labeta (average of 6 measurements for each rat together with - ZEM).

Cumulative cold score, defined as the sum of 6 points for each rat together x ZEM.

The minimum score is 0 (no response on each of the b trials), and the maximum possible score is 18 (repeated jerking and licking or biting of the labia, on each of the six trials). because Statistical analysis

Student's test, one-sided, type C was performed. The significant level was set as p « 0.05; all groups were compared with the carrier group (group treated with oxaliplatin).

Means and rms error of the mean are shown in the figures.

The results

Oxaliplatin induces a significant decrease in the reaction time of the labeta withdrawal after application of acetone (sick group "t carriers") in a time-dependent manner. This reduction is progressive and significant from day 1 (acute model of oxaliplatin-induced neuropathy) to day 8 (chronic model) compared to the vehicle group. - Antiallodynic effect in an acute model of oxaliplatin-induced neuropathy

Drug combinations tested in an acute model of oxaliplatin-induced neuropathy were evaluated using the acetone test. Table 12 presents the drug combination (Group 4) that induces a significant decrease in the cumulative cold score and a significant decrease in reaction time compared to the vehicle oxaliplatin-treated group (Group 2). In conclusion, it should be noted that these drug combinations protect animals from acute neuropathy induced by oxaliplatin.

Table 12

Combinations of medicinal products Variation of cold Reaction time in Antiallodynic model of acute neuropathy score in comparison with effect (Group 4) with Group 2 group 2 baclofen-torasemide decrease increase no baclofen-acamprosate- decrease increase no torasemide decrease increase no mexiletine and cinacalcet decrease increase no sulfisoxazole and torasemide - same antiallodynic effect obtained in Group C rats, after analysis of cumulative cold scores and analysis of reaction time in acetone tests, in an acute oxaliplatin-induced model. - Antiallodynic effect in a chronic model of oxaliplatin-induced neuropathy

Drug combinations used in a chronic model of oxaliplatin-induced neuropathy were evaluated using the acetone test.

Table 13 presents drug combinations for which reaction time and acetone cold score measured in Group 4 (animals treated with combinations of drugs and oxaliplatin), respectively, are significantly increased and decreased after treatment in a model of chronic neuropathy in compared with the group treated with vehicle oxaliplatin (Group 2). In conclusion, it should be noted that these drug combinations protect animals from oxaliplatin-induced chronic neuropathy.

Table 13

Combinations of drugs, Variation of cold Reaction time in Antiallo- tested in the chronic model of the score in comparison with comparison with dynamic neuropathy (Group 4) Group 2 group 2 effect baclofen-torasemide decrease increase nia baclofen-acamprosate-torasemid decrease increase nia mexiletine and cinacalcet decrease increase in sulfisoxazole and torasemide decrease increase in C - the same antiallodynic effect obtained in Group C rats, after analysis of cumulative cold scores and analysis of reaction time in acetone tests, in the chronic oxaliplatin-induced model.

IM) Compositions based on baclofen-torasemide stimulate nerve regeneration in non-intoxicated cells - Neurotrophic effects of baclofen-torasemide combination ip migo

Neurite length test

Evaluation of neurite growth in 10-day cultures of rat cortical cells was carried out using MAP-2 antibodies as mentioned in section A) II.4, except that the cells were not exposed to any toxic substances. 10-day-old cell cultures were incubated with drugs for 1 day before the test.

The results

As shown in Figure 28, the combination of baclofen-torasemide shows a significant neurotrophic effect (111 95), whereas the individual drugs, when used alone, do not show any significant neurotrophic effect. Indeed, a significant increase in the total length of neurites within the neuronal network (marked by MAP2-2) is observed upon exposure to the combination of baclofen and torasemide (400 nM and 80 nM, respectively). Note that neither the combination nor the drugs alone have any effect on the number of neurons, thus emphasizing that this increase in the neurite network is associated with the elongation of existing neurites and de pomo stimulation of neuronal cell elongation.

These results confirm that the baclofen-torasemide combination is effective in supporting axonal elongation and thus effective in the treatment of spinal cord and other nerve lesions. This also confirms that the combination is effective in the treatment of hereditary neuropathies that confirm either an axonal or a demyelinating component, or both an axonal and a demyelinating component. Indeed, it should be considered that demyelination causes axon destabilization, leading to axonal degeneration, which is observed even in neuropathies that are considered to be mainly demyelinating. - Compositions based on baclofen-torasemide are effective in activating the regeneration of nerves of the ip

By the way

Sciatic nerve compression is widely accepted as a valid model of peripheral nerve injury and for assessing nerve regeneration. In this model, nerve injury leads to rapid destruction of nerve function, as evidenced by a measure of evoked muscle action potential (EMP) generated by stimulation of the affected sciatic nerve.

Damage to the nerve is characterized by a lower conduction of the signal by the nerve, which leads to an increase in the delay in the formation of SMAR and a disturbed strength of the action potential, which gives a reduced amplitude and duration.

Typically, the first signs of recovery of nerve function occur within 2 weeks, and by week 4 post-injury, significant remyelination of regenerated axons is observed in the sciatic nerve by histology (45).

Nerve compression

Mice were anesthetized with isoflurane (2.5-3 95 in air). The right thigh was then shaved and the sciatic nerve was exposed at mid-thigh level and compressed 5 mm proximal to the bifurcation of the sciatic nerve. The nerve was compressed for 10 s twice using microclamps (NockKech,

hetegepse RZ5311) with 907 turns between each squeeze. For sham-operated animals, the sciatic nerves were exposed but not compressed. Finally, the skin incision was secured with wound clips. The day of squeezing is treated as day 0.

Dosage schedule

The day of compression, the first introduction of components was carried out 30 minutes after compression.

The compounds were then administered twice a day, starting the day after compression and for 42 days. Within one day, administration of medicines were separated by an interval of at least 6 hours.

On the test days (days 7 and 30), mice were injected for 1 hour. 30 min. before the test.

The volume of administration was 10 ml/kg, in 0.2595 DMSO/sterile water. 11111111 | Dose! (rb) | Dosa2(genus | DozaZ(fid

Measurement of electromyography

Recording of electrophysiological data was carried out using a Keuroipy electromyograph (EMO) (Meaigopis, France) on day 7 and day 30. Mice were anesthetized with 2.5-3 9o isoflurane in air. Subcutaneous monopolar needle electrodes were used for both stimulation and recording. Supramaximal (12.8 mA) rectangular pulses with a duration of 0.2 ms were used to stimulate the sciatic nerve. The right sciatic nerve (ipsilateral) was stimulated with a single pulse applied to the sciatic notch. SMAR was recorded with needle electrodes placed on the leg muscles. The onset (delay) of the SMAR signal, expressed in milliseconds, was used to estimate the nerve conduction velocity. The delay thus reflects the degree of myelination of axons. Action potential amplitude (AP) was also determined, which reflects the level of muscle denervation and reinnervation. The amplitude of SMAR at this time is given as proportional to the number of regenerated motor axons. The duration of the evoked muscle potential was also determined. The amplitude and duration of the evoked muscle potential is more related to muscle reinnervation. Latency and amplitude are commonly recognized as the most important endpoints in the case of nerve regeneration. The data were analyzed using a bilateral, C-type, Student's Y-criterion; a significant level is established at p«0.05.

The results

The baclofen-torasemide combination is effective at all doses tested, with significantly improved signal latency compared to vehicle-treated animals, as early as day seven after nerve injury (FIGURE 29, A). A significant difference is still observed at day 30 after nerve compression, when spontaneous recovery usually begins. 35 A statistically significant improvement in signal amplitude was also observed for Dose C on the day after nerve compression (Figure 29, B). This improvement is also observed, but to a lesser extent, for doses 1 and 2. Similar results are observed when measuring signal duration.

Overall, these ip mimo results demonstrate the efficacy of the baclofen-torasemide combination in stimulating nerve regeneration through remyelination and muscle reinnervation. Therefore, experimental data confirm the neurotrophic effects of baclofen-torasemide observed ip and their usefulness in the correction of neuropathies when the nerves of the peripheral nervous system are damaged (i.e., the neuropathies defined in the description), but also in spinal cord lesions.

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Claims (1)

FORMULA OF THE INVENTION 1. A combination comprising at least torasemide and baclofen or salts thereof for the regeneration of nerves or neurons in a subject suffering from nerve damage selected from neurotaxis, axonotmesis or neurotmesis, neuropathy caused by direct physical insult to the nerve or Charcot disease Marie-Tuta. 2. The combination according to claim 1, which is characterized by the fact that it includes at least one compound selected from sulfisoxazole, methimazole, prilocaine, diphylline, quinacrine, carbenoxolone, acamprosate, aminocaproic acid, baclofen, cabergoline, diethylcarbamazine, cinacalcet, cinnarizine, eplerenone, fenoldopam , leflunomide, levosimendan, sulodexide, terbinafine, zonisamide, etomidate, phenformin, trimetazidine, mexiletine, ifenprodil, moxifloxacin or bromocriptine, or their salts. 3. The combination according to claim 2, which differs in that it includes at least one of the following combinations of compounds: - baclofen, trimetazidine and torasemide, - baclofen, cinacalcet and torasemide, - baclofen, acamprosate and torasemide, - baclofen, acamprosate and torasemide and diethylcarbamazine , or - baclofen, acamprosate and torasemide and ifenprodil. 4. The combination according to any of the previous items, which is characterized by the fact that it includes a pharmaceutically acceptable carrier or excipient. 5. A combination according to any of the previous items, characterized in that the compounds are formulated for administration together, separately or sequentially. 6. The combination according to any of the previous items, which differs in that the compounds are administered to the subject repeatedly. 7. The combination according to any of the preceding items, which is characterized in that it includes less than 4 mg of torasemide. 8. The combination according to any one of the preceding items, which is characterized by the fact that it includes less than 150 mg of baclofen, preferably less than 50 mg. 9. A method of stimulating nerve or neuron regeneration in a subject suffering from nerve damage selected from neurotaxis, axonotmesis, neurotmesis, neuropathy caused by direct physical insult to the nerve or Charcot-Marie-Tooth disease, which involves administration to said subject torasemide and baclofen or their salts. 10. The method according to claim 9, which is characterized by the fact that it additionally provides for the introduction of a pharmaceutically acceptable carrier or filler. 11. The method according to claim 9, which differs in that it involves simultaneous, separate or sequential administration of torasemide and baclofen to the subject. 12. The method according to claim 9, which differs in that it provides for the introduction of less than 4 mg of torasemide. 13. 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Y : m: she - BA p | MOM od dnshimivmy SCHO A Georges g : I : 5" veze : t E KOVAH : I o" and ge MO E I : Z OO I sce i 8 Po: I and N be "lety IS KOH, : . kE them OO not peOzhe " KU peaks. Hay zo VOVYKH NI : not zho and YAKA izh : control glue kZh zva) pu He : chom z Vaclofenoyai ZM Ge : y zhomeksnletin SUYA AM o. zh SO : B: o ZV ! : si NO Ka KTK, vasuroati igsyu ET she, shu tikh ekrvliv LYNOTI and PM More Fig. 8 zhk eat : 8 EY and poh : A and p- B x 8 kozh zv customs | r : o : : i poh sl same them that I in ru VIDOVA. I : M m ME : for | in ! pe : I sho KO E gav po OO this OV N schu MDU Y M -k 4 ha hm pe sho gos pt 1 moni l ke y: shrot x : i Tezh CY GB : ХЕ i 7 i Yu zh i , . : - зв и г 5 : x |. ше что у 5 Го ЖК ково и: и Я я х Ох: рОщ КЗ пы ш E: ate: - ОО ож. 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I MOH is still OK: in the SAME. ше ОО b» что 5 - о ши ШЭ Х х on | h a Z OH N oyu OKOMKIE: well NO and her oo: Z Ko i | what B MNK! ShMK gays KEKKya Z . N ze SELOM 2 OK ZU 4 pnia ' stpihuyetu zhi S KOH Ky : : ! Trollu as: schi MO U - Y 18 u ma ME those that M E opti m: saw hosts New xx EK pek tu nn nt yuyu: Ktegbnnets s me DU VOK eV. nan i te te KN U : i 56 OK i N VENU I Her Koch. OO HE i what YOU Oya no nya JHE Zk KE OH : E t.e what hmm ovE v y : ! that IOYKEOE S ego I what: their PE r - dekhh oh yozi them: po I klem z luk amya: highway SEBOH koteranatsfia BO mKM still Ж B posle - V ia tToracenis VE NM E is B VO p y hol OH z PEMMh x Med And that is MAM. : en her ME s ZoNNN p sho PSHU SAKO "I: stelyakh p proyu invernh ; and condyloma Fig. 13 AXES that Zh r x A pk shemevaya V «va . ? i PO te KK oo S ECO ee BUT WAK ZHrya SYA - MA Z, Ж Be ХХ s EE ro. 00 O koh o shob STEKUMOV o SKK Ah Z sh-h MJUVANE Z Ya re de po POS - Zh Me U I p: OKO p x p i von BOYU ney Z OK | - r SK yav AB | in 0 ZK : : OO VOM ky I : Ou pe MOZHU ia KSO pov Fe 0 MOB Y che M a 5 y BEBI . 38 OH MY Y Shi OH 5 ov and povo JUKOM ZH KO : : : CHO dedunny j COKO o x S syad N ya LIKMY: me . MO Xia h i she . U kov still and i dom tk "Bo MM M dela: and picdnomymmA ke M V BUH U plex zhi I KOSYM - 1 But OV KOL as ZM NYA shchi SCHE - VO Bint og KK x vin I skh: KM KVU i a PLAN km N BU KO : : yah pooh "VN : Ko SHY S kovhrov what! in : tsk kuki there | IV pyzha « stump and in her 7 KO Ya mmupakogvy m Ya t Zh PIzheh, y zi i eh Iii PV esashktri mno - What DAK Pa I Zh EE sia Zh x I IDVANNE - PINA «E KYN. Isaiah 5. PO VE MUMIYSN" . : what a house of those " And G. zostohEshuikis and i im ha Uh tse e detttyatteny yana la vdl ih jutyuini tp Z ov | po, Pt : - Because o : : what you would have : : for dove ! ota": ! : for V | ry ! : ki SA -4 N : " va ie i ! KO and WE and : : PO sho : : : y : 70 B : : WE Y : Й : KOKO spilled With m : : : OK willingly g : : : vo OO I VEU nyah N I : Kantrope Ave KAK aa; zon demo: Kaya Alesv is liu: - and still kot rn Dr, I Sa still I en Baklenemo Yaog niy odkiskden IAM udrasyumid we not zhk terasamid jan 1 5 Fig 15 eV as And her What . 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And 1 1 that MO - : - : KU PO t ! oh SKK : and that's OK, Z Moh se MKK | pi more -y YAKU OKH : Y , and OK not and 1 she VO A Ne | p mn OO Beru" and Shchi ECO ZK 7 E N I But and koktrol' that Avmid io y : dao vva: nvK - VOYU : there are mahkyfizkovlynYA MM ih v y Y laku i YN z i levimetazidin; ZoY NM sho MKK Ziniv 3 4X actions PM. soyu ZVT Wu ED z cha Fig 17 - shkh ny x Y 7 Pou yiv i ; В Е д : шо - Ша Ж ж y y : uinnvnavuyu we you -5- kh kemeKee : them fo Y ko OO I 1 same with her. : and "ok I'm MUK; tai pov KO : ; t OM Oh What! take off two o'clock some mice : | between the ages of KOM we found ov; ше I шчеоВ: as ВХ BK kn i: Z zzazhoyaripldin Z Am KK OKH i ! tya she and N : I 5 - OO Toy : I: Z « V I gm : Fo , and KK OO . N More: I y Y y i Ya ke: zho and MZHK tlia x x Z z KOKO | lot | y : : Koral deya 1 via3z42. zby I Mo and Neo kmevochrnlyusevii aa and their myny "lov umimih VO NAI o p I z zu MEM t NK OKH Y ! ABNOKH t VE : OPO : still Y Zdna 14 VI YAM boom FNg 1 Z SHE memories Z y ' ' their t Y x mu i V to A Zk - ! to not i-th 1 | : : Mo Zh ikh : y Chho - 7 V ie pl OO phkokah : 1 in Fo Z x LEO I g. in we, x BM I ho How is s : : і шішн ! about ZE! m" Vkva: any dust | seh BMK Ku pi AD pd 4 p. ni shsh KayairOl VVE. PTO PTO FOR RIDING | ТА ЗПИЙА дезрчжнплвы ДИМ ху ВОВХХ more! : LANGUAGES of NM NGO Dogs! in and | what oh! i S EM Sm a - ! E p An i V M I PT i i i | | EZNVI: o I NYA N Osy. p: ate Shk "5 M UNO 3 s I Pray they, control AVI-42 in the ear | khy luky sv i y : shcha session sh zu siya g Shk KO VE "Vnaltse tya pai j what VOK : Z OKH "8 V FI g 1 Od honlol AVEZUH vicha im I N yak kah, VN upinu v: VH B p i z Neo « , В A bo: : іт що мю щой Го : : куме Я и а це Я a Ж N ta : Z - N tsk dova : beer tributaries lk i ry p : Y POKO : : ih i OO is DYN | z Zh : : z 4 : : --y TEM : Z: ! I KAB MO nevohuya and t: HYNTROL oAVBLU slrBya) p BE iy | | bom ke I : sha: ! shk ; I ; s : : and what 5 I mohy iovynoya kiy VO ko ovy : : to eat shchi oh : | m ha EE " E : i o I p : zol ; - i i | Stiles : as where For. IT: N no KI opo) m Ton zo V vl ya " z oo o M ShK 1 pe o: KKU I ET dvemaeny shi Vi ! | on eyes ME control two 0 Ah DVZHM is she she : V i, BU KE M TAM 0 zokhzhoyuvoyutyuh I YAM x B !! deniramid; Z Km - EV p. I SEA pi yatrove o AvVi-42 ABI I" pam. fi 2 0 Tamoz zoveamid: Zav IIE niya I what Y y. V AD - - dra m pray more Я о. KESHTYA i as those p same : - show some more ve OKO I : y : t M kn v NI COKO :. 1 re S OH ; : : i AY - is VOM : sht ti i pe -. OK : і : de : " VOMU : : ! more : CONT KBet 45 y KA x M : would 00 every Yukratsyan NM shodo t shtokom ley y KUZYA szhpyufrIshvko im tat U: still ho BE : sho sko 0 PAOZHYMYKH khanino dez I VIYAMI SHE v. ark j WHOSE OVIOgO p Fig. 21 1804. i Y same F 60 shk she ; iz SCHE zho " 44 more Ж 140 : p. to : | is AI: I o in 1 No 0 CENTURY Se AI. ve o o VED of the day --.. ZY nya kisntrnnnya i M Ko i Ge 5" zhi "KA s E x ky Ж ki o ih x Baga - x Kz x u -b y x i Kk 4 - Ye I Be 2 ; Ol k still s shcha. still x t 4 F Fig. 22 with a. 2004 - o - 175th school and 195th school! and. M 8 0 veto her about with Ya scha OR! re ko Dogomt i glorious lina. chn pe - dnk as Yam Shh - Be still more Va. yati tob ve B t iya She Ya t-boO ZO x zhk V Ou Ize) : y xx nei ke : ! I have Zoe. VV. . OVT ACCOUNT; cycles of Russian-made KALIZHKT Kuk that tuto ovezh Mo i her Okht z -o 40 x Ne o : i : E zak a za. Ж п ше 7 у» M pa mother village and types of growth, i SELOM VN tata ud M zh mil lIMID Fig. 25 х 0 х 120 х Х Ж and их ше and в. Dya Bo EE KK sa MO V v, i still teh is o o U Ka i schu ai o ai K et i Fig. 26 and 68: And oh no S ziao! - Mr. What x x F y Ne Z Z vu ; with: e zay i: E povo | ak y I OO A VA B She : ! : J 5. And ! : Ge ХВ ОО PINSHE : : : I s son, EK VOK KE and i : that POZ and I : more KO about them : poni : yati z" vv VE , | shi : and control Arega 12 Abeta ibnAya dvez M Hor ktomotauevin, It is shown in Fig. 2 and 27 115. : si yaku dleryaushchy poyu: pi « ; pek - CURRENT pe KOSI: TEH pu: w 305. B -: Z to F: PE! ge and y she r : oh, ! - : MOSS Oh WOMAN! her o I control Baclofen (400 nm baclofen (I yami yatorasemide (80 NIM) that Schi torasemide (80 nimi) Fig. 28